U.S. patent number 6,515,113 [Application Number 09/507,630] was granted by the patent office on 2003-02-04 for phthalamide lanthanide complexes for use as luminescent markers.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Seth M. Cohen, Stephane Petoud, Kenneth N. Raymond, Jide Xu.
United States Patent |
6,515,113 |
Raymond , et al. |
February 4, 2003 |
**Please see images for:
( Certificate of Correction ) ** |
Phthalamide lanthanide complexes for use as luminescent markers
Abstract
The present invention provides luminescent lanthanide metal
chelates comprising a metal ion of the lanthanide series and a
complexing agent comprising at least one phthalamidyl moiety. Also
provided are probes incorporating the phthalamidyl ligands of the
invention and methods utilizing the ligands of the invention and
probes comprising the ligands of the invention.
Inventors: |
Raymond; Kenneth N. (Berkeley,
CA), Petoud; Stephane (Berkeley, CA), Cohen; Seth M.
(Boston, MA), Xu; Jide (Berkeley, CA) |
Assignee: |
The Regents of the University of
California (Oakland, CA)
|
Family
ID: |
22393079 |
Appl.
No.: |
09/507,630 |
Filed: |
February 18, 2000 |
Current U.S.
Class: |
534/15; 435/4;
435/6.12; 436/546; 436/804; 534/16 |
Current CPC
Class: |
A61K
41/0057 (20130101); A61K 49/0002 (20130101); A61K
49/0019 (20130101); A61K 49/103 (20130101); A61K
49/106 (20130101); A61K 49/122 (20130101); A61K
49/124 (20130101); A61K 51/0478 (20130101); A61K
51/0482 (20130101); A61K 51/06 (20130101); C07C
235/60 (20130101); C07D 487/08 (20130101); C07D
487/18 (20130101); C07D 487/22 (20130101); C09K
11/06 (20130101); C12Q 1/37 (20130101); G01N
33/582 (20130101); C09K 2211/1007 (20130101); C09K
2211/182 (20130101); G01N 2458/40 (20130101); Y10S
436/80 (20130101); Y10S 436/804 (20130101) |
Current International
Class: |
C07C
235/60 (20060101); C07C 235/00 (20060101); C07F
5/00 (20060101); C07D 487/08 (20060101); C07D
487/00 (20060101); C07D 487/18 (20060101); C07D
487/22 (20060101); C09K 11/06 (20060101); C12Q
1/25 (20060101); C12Q 1/68 (20060101); C12M
1/00 (20060101); C12N 15/09 (20060101); G01N
33/566 (20060101); G01N 37/00 (20060101); G01N
33/53 (20060101); G01N 33/533 (20060101); C07F
005/00 (); G01N 033/533 () |
Field of
Search: |
;534/15,16 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
5049280 |
September 1991 |
Raymond |
5820849 |
October 1998 |
Schmitt-Willich et al. |
|
Foreign Patent Documents
|
|
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|
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|
2099542 |
|
Jul 1993 |
|
CA |
|
0 578 067 |
|
Jan 1994 |
|
EP |
|
WO 92/11039 |
|
Jul 1992 |
|
WO |
|
Other References
Blomberg, et al., "Terbium and rhodamine as labels in a homogeneous
time-resolved fluorometric energy transfer assay of the .beta.
subunit of human chorionic gonadotropin in serum", Clinical
Chemistry, 45(6):855-861 (1999). .
Bunzli, et al., "Towards materials with planned properties:
dinuclear f-f helicates and d-f non-covalent podates based on
benzimidazole-pyridine binding units", Journal of Alloys and
Compounds, 249:14-24 (1997). .
Chen, et al., "Lifetime- and color-tailored fluorophores in the
micro- to millisecond time regime", J. Am. Chem. Soc.,
122(4):657-660 (2000). .
Dickins, et al., "Synthesis, time-resolved luminescence, NMR
spectroscopy, circular dichroism and circularly polarised
luminescence studies of enantiopure macrocyclic lanthanide
tetraamide complexes", Chem. Eur. J., 5(3):1095-1105 (1999). .
Dickson, et al., "Time-resolved detection of lanthanide
luminescence for ultrasensitive bioanalytical assays", Journal of
Photochemistry and Photobiology, B:Biology, 27:3-19 (1995). .
Galaup, et al., "Mono(di)nuclear europium(III) complexes of
macrobi(tri)cyclic cryptands derived from diazatetralactams as
luminophores in aqueous solution", Helvetica Chimica Acta,
82:543-560 (1999). .
Hemmila, et al., "Development of luminescent lanthanide chelate
labels for diagnostic assays", Journal of Alloys and Compounds,
249:158-162 (1997). .
de Sa, et al., "Spectroscopic properties and design of highly
luminescent lanthanide coordination complexes", Coordination
Chemistry Reviews, 196:165-195 (2000). .
Sabbatini, et al., "Luminescent lanthanide complexes as
photochemical supramolecular devices", Coordination Chemistry
Reviews, 123:201-228 (1993). .
Saha, et al., "Time-resolved fluorescence of a new europium chelate
complex: Demonstration of highly sensitive detection of protein and
DNA samples", J. Am. Chem. Soc., 115:11032-11033 (1993). .
Soini, et al., "Time-resolved fluorescence of lanthanide probes and
applications in biotechnology", CRC Critical Reviews in Analytical
Chemistry, 18(2):105-154 (1987). .
Steemers, et al., "Water-soluble neutral calix[4]arene-lanthanide
complexes: Synthesis and luminescence properties", J. Org. Chem.,
62:4229-4235 (1997). .
Stenroos, et al., "Homogeneous time-resolved IL-2IL-2R.alpha. assay
using fluorescence resonance energy transfer", Cytokine,
10(7):495-499 (Jul., 1998). .
Veiopoulou, et al., "Comparative study of fluorescent ternary
terbium complexes. Application in enzyme amplified fluorimetric
immunoassay for .alpha.-fetoprotein", Analytica Chimica Acta,
335:177-184 (1996). .
Vicentini, et al., "Luminesence and structure of europium
compounds", Coordination Chemistry Reviews, 196:353-382
(2000)..
|
Primary Examiner: Ceperley; Mary E.
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Government Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
This work was partially supported by grants from the National
Institutes of Health (DK32999) and the United States Department of
Energy (DEAC0376F00098). The Government may have rights in the
subject matter disclosed herein.
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent
Application Serial No. 60/120,881, filed on Feb. 18, 1999. This
application is also related to U.S. Provisional Patent Application
Serial No. 60/120,600, filed on Feb. 18, 1999 and U.S. patent
application Ser. No. 09/507,599, and filed on an even date
herewith. Each of these documents are incorporated herein by
reference in their entirety for all purposes.
Claims
What is claimed is:
1. A luminescent lanthanide metal chelate comprising a metal ion of
the lanthanide series and a complexing agent comprising at least
one moiety derived from 2-hydroxy-1,3-benzenedicarboxylic acid.
2. The chelate according to claim 1, having a quantum yield of at
least about 0.1.
3. The chelate according to claim 2, wherein said lanthanide metal
ion is a member selected from europium, terbium and combinations
thereof.
4. The chelate according to claim 1, further comprising at least
one salicylamidyl moiety.
5. A luminescent lanthanide metal chelate according to claim 1
wherein said at least one moiety derived from
2-hydroxy-1,3-benzenedicarboxylic acid has the formula:
##STR23##
in which R.sup.1, and R.sup.2 are members independently selected
from the group consisting of H, alkyl and substituted alkyl groups;
R.sup.11, R.sup.12, and R.sup.13 are members independently selected
from alkyl, substituted alkyl, H, --NR.sup.14 R.sup.15, --NO.sub.2,
--OR.sup.16, --COOR.sup.17, wherein, R.sup.14, R.sup.15, R.sup.16
and R.sup.17 are members independently selected from the group
consisting of H, alkyl and substituted alkyl, wherein R.sup.12 can
optionally form a, ring with R.sup.11, R.sup.13 or both, said ring
being a member independently selected from the group of ring
systems consisting of cyclic alkyl, substituted cyclic alkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl
and saturated heterocyclyl ring systems; and Q.sup.1 is --R.sup.18
; wherein R.sup.18 is selected from H, an enzmatically labile
group, a hydrolytically labile group and a single negative charge.
Description
BACKGROUND OF THE INVENTION
There is a continuous and expanding need for rapid, highly specific
methods of detecting and quantifying chemical, biochemical and
biological substances as analytes in research and diagnostic
mixtures. Of particular value are methods for measuring small
quantities of nucleic acids, peptides, pharmaceuticals,
metabolites, microorganisms and other materials of diagnostic
value. Examples of such materials include small molecular bioactive
materials (e.g., narcotics and poisons, drugs administered for
therapeutic purposes, hormones), pathogenic microorganisms and
viruses, antibodies, and enzymes and nucleic acids, particularly
those implicated in disease states.
The presence of a particular analyte can often be determined by
binding methods that exploit the high degree of specificity, which
characterizes many biochemical and biological systems. Frequently
used methods are based on, for example, antigen-antibody systems,
nucleic acid hybridization techniques, and protein-ligand systems.
In these methods, the existence of a complex of diagnostic value is
typically indicated by the presence or absence of an observable
"label" which has been attached to one or more of the interacting
materials. The specific labeling method chosen often dictates the
usefulness and versatility of a particular system for detecting an
analyte of interest. Preferred labels are inexpensive, safe, and
capable of being attached efficiently to a wide variety of
chemical, biochemical, and biological materials without
significantly altering the important binding characteristics of
those materials. The label should give a highly characteristic
signal, and should be rarely, and preferably never, found in
nature. The label should be stable and detectable in aqueous
systems over periods of time ranging up to months. Detection of the
label is preferably rapid, sensitive, and reproducible without the
need for expensive, specialized facilities or the need for special
precautions to protect personnel. Quantification of the label is
preferably relatively independent of variables such as temperature
and the composition of the mixture to be assayed.
A wide variety of labels have been developed, each with particular
advantages and disadvantages. For example, radioactive labels are
quite versatile, and can be detected at very low concentrations,
such labels are, however, expensive, hazardous, and their use
requires sophisticated equipment and trained personnel. Thus, there
is wide interest in non-radioactive labels, particularly in labels
that are observable by spectrophotometric, spin resonance, and
luminescence techniques, and reactive materials, such as enzymes
that produce such molecules.
Labels that are detectable using fluorescence spectroscopy are of
particular interest, because of the large number of such labels
that are known in the art. Moreover, the literature is replete with
syntheses of fluorescent labels that are derivatized to allow their
facile attachment to other molecules, and many such fluorescent
labels are commercially available.
In addition to being directly detected, many fluorescent labels
operate to quench the fluorescence of an adjacent second
fluorescent label. Because of its dependence on the distance and
the magnitude of the interaction between the quencher and the
fluorophore, the quenching of a fluorescent species provides a
sensitive probe of molecular conformation and binding, or other,
interactions. An excellent example of the use of fluorescent
reporter quencher pairs is found in the detection and analysis of
nucleic acids.
An alternative detection scheme, which is theoretically more
sensitive than autoradiography, is time-resolved fluorimetry.
According to this method, a chelated lanthanide metal with a long
radiative lifetime is attached to a molecule of interest. Pulsed
excitation combined with a gated detection system allows for
effective discrimination against short-lived background emission.
For example, using this approach, the detection and quantification
of DNA hybrids via an europium-labeled antibody has been
demonstrated (Syvanen et al., Nucleic Acids Research 14: 1017-1028
(1986)). In addition, biotinylated DNA was measured in microtiter
wells using Eu-labeled strepavidin (Dahlen, Anal. Biochem, 164:
78-83 (1982)). A disadvantage, however, of these types of assays is
that the label must be washed from the probe and its fluorescence
developed in an enhancement solution. A further drawback has been
the fact that the fluorescence produced has only been in the
nanosecond (ns) range, a generally unacceptably short period for
adequate detection of the labeled molecules and for discrimination
from background fluorescence.
In view of the predictable practical advantages it has been
generally desired that the lanthanide chelates employed should
exhibit a delayed fluorescence with decay times of more than 10
.mu.s. The fluorescence of many of the known fluorescent chelates
tends to be inhibited by water. As water is generally present in an
assay, particularly an immunoassay system, lanthanide complexes
that undergo inhibition of fluorescence in the presence of water
are viewed as somewhat unfavorable or impractical for many
applications. Moreover, the short fluorescence decay times is
considered a disadvantage of these compounds. This inhibition is
due to the affinity of the lanthanide ions for coordinating water
molecules. When the lanthanide ion has coordinated water molecules,
the absorbed light energy (excitation energy) is transferred from
the complex to the solvent rather than being emitted as
fluorescence.
Thus, lanthanide chelates, particularly coordinatively saturated
chelates having excellent fluorescence properties are highly
desirable. In the alternative, coordinatively unsaturated
lanthanide chelates that exhibit acceptable fluorescence in the
presence of water are also advantageous. Such chelates that are
derivatized to allow their conjugation to one or more components of
an assay, find use in a range of different assay formats. The
present invention provides these and other such compounds and
assays using these compounds.
SUMMARY OF THE INVENTION
Luminescent (including fluorescent and phosphorescent) markers find
a wide variety of applications in science, medicine and
engineering. In many situations, these markers provide competitive
replacements for radiolabels, chromogens, radiation-dense dyes,
etc. Moreover, improvements in fluorimetric instrumentation have
increased attainable sensitivities and permitted quantitative
analysis.
Lanthanide chelates in combination with time-resolved fluorescent
spectroscopy is a generally accepted immunochemical tool. Presently
preferred lanthanide ions include Dy.sup.3+, Sm.sup.3+, Tb.sup.3+,
Er.sup.3+ and Eu.sup.3+, Nd.sup.3+, Yb3+. Other lanthanide ions,
such as La.sup.3+, Gd.sup.3 + and Lu.sup.3 + are useful, but
generally less preferred.
The present invention provides lanthanide complexes that are
extremely luminescent and possess many features desired for
fluorescent markers and probes of use in fluorescent assay systems.
Among these advantages are: 1) ligands acting as both chelators and
chromophore/energy transfer devices; 2) very high quantum yields of
lanthanide ion fluorescence of the present complexes in water
without external augmentation, such as by micelles or fluoride; 3)
high stability and solubility of these complexes in water; 4) an
extremely easy synthesis that employs inexpensive starting
materials; and 5) facile access to many derivatives for linking
these luminescent probes to, for example, an immunoreactive agent
or solid support (e.g., polymer).
The present invention provides a new class of lanthanide-complexing
ligands that incorporate hydroxyisophthamidylic acid moieties
within their structures and luminescent metal complexes of these
ligands. The compounds of the invention include
hydroxyisophthamidylamide-based bidentate, tetradentate and other
higher polydentate ligands. The compounds of the invention are
easily prepared in good yields.
Thus, in a first aspect, the present invention provides a
luminescent lanthanide metal chelate comprising a metal ion of the
lanthanide series and a complexing agent comprising at least one
phthalamidyl moiety.
In a second aspect, the invention provides a compound having a
structure according to Formula I: ##STR1##
In Formula I, R.sup.1, R.sup.2, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.10 and R.sup.20 are members independently selected from the
group consisting of H, alkyl and substituted alkyl groups, wherein,
two or more of R.sup.2, R.sup.4, R.sup.5, R.sup.7 and, when R.sup.3
is substituted alkyl, a substituent of R.sup.3 are optionally
adjoined by at least one linker moiety to form at least one ring.
R.sup.3, R.sup.8 and R.sup.9 are members independently selected
from the group consisting of alkyl, substituted alkyl, aryl and
substituted aryl groups. R.sup.11, R.sup.12, R.sup.13, R.sup.21,
R.sup.22 and R.sup.23 are members independently selected from
alkyl, substituted alkyl, H, --NR.sup.14 R.sup.15, --NO.sub.2,
--OR.sup.16, --COOR.sup.17, wherein, R.sup.14, R.sup.15, R.sup.16
and R.sup.17 are members independently selected from the group
consisting of H, alkyl and substituted alkyl, wherein R.sup.12 can
optionally form a ring with R.sup.11, R.sup.13 or both, and
R.sup.22 can optionally form a ring with R.sup.21, R.sup.23 or
both. The rings are members independently selected from the group
of ring systems consisting of cyclic alkyl, substituted cyclic
alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclyl and saturated heterocyclyl ring systems. Q.sup.1 is
--OR.sup.18 and Q.sup.2 is --OR.sup.19, wherein R.sup.18 and
R.sup.19 are members independently selected from H, an
enzymatically labile group, a hydrolytically labile group and a
single negative charge. The letter a is 0 or 1, with the proviso
that when a is 0, N.sup.2' is covalently attached directly to
carbonyl group 2' and z is 0 or 1, with the proviso that when z is
0, N.sup.1' is covalently attached directly to carbonyl group
1'.
In addition to the ligands and lanthanide complexes, the present
invention also provides a number of methods, including assays
utilizing the compounds of the invention. The assays of the
invention preferably utilize the fluorescence of the compounds
described herein to detect the subject of the assay. The methods of
the invention allow the detection of, for example, small molecular
bioactive materials and biomolecules at trace concentrations
without using radioactive species.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an exemplary synthetic scheme leading to the ligand
H.sub.3 (bicapped TRENSAM).2HBr
FIGS. 2(A-B) are experimental (A) and calculated (B) spectra from
the titration of bicapped TRENSAM with EuCl.sub.3 in buffered
aqueous solution. In FIG. 2A, spectra 1 to 18 correspond to
increasing concentrations of lanthanide ion. In FIG. 2B, the solid
line is bicapped TRENSAM, the dotted line is Eu[bicapped
TRENSAM].sub.2, and the dashed line is Eu[bicapped TRENSAM].
FIG. 3 is an overlay plot of an experimental electronic spectrum
(solid line) and calculated electronic spectrum (dotted line) of
Eu[bicappedTRENSAM].sub.2.
FIGS. 4(A-B) are structural diagrams, ORTEP (A) at 20% probability
ellipsoids and space-filling (B), for [Eu(bicappedTRENSAM).sub.2
].sup.+. Hydrogens, solvent, and counterion are omitted for
clarity.
FIGS. 5(A-B) are structural diagrams (ORTEP) of the metal center in
[Eu(bicappedTRENSAM).sub.2 ].sup.+. The metal center coordination
polyhedron is a slightly distorted square antiprism (A) with each
face of the antiprism formed by one of the bicapped TRENAMSAM
ligands (B). 50% probability ellipsoids shown.
FIG. 6 is an overlay plot of emission spectra of free bicapped
TRENSAM (solid line, 4.24.multidot.10.sup.-4 M in water (Millipore
purified)), of [Tb(bicappedTRENSAM).sub.2 ].sup.+ (dashed line,
1.3.multidot.10.sup.-5 M in water (Millipore purified)) and of
[Eu(bicappedTRENSAM).sub.2 ].sup.+ (dotted line,
1.08.multidot.10.sup.-4 M in water (Millipore purified)) with 0.1 M
KCl, 0.05 MES buffer, adjusted to pH 5.78 with KOH.
FIGS. 7(A-B) are structural Formulae of representative dendrimers
of use in the present invention.
FIG. 8 is a representative synthetic scheme leading to an exemplary
tetrapodal ligand of the invention.
FIG. 9 is a representative synthetic scheme leading to an exemplary
compound of the invention derivatized with a linker arm providing a
locus for attaching the ligand to another species.
FIG. 10 is a representative synthetic scheme leading to exemplary
compounds of the invention derivatized with alkyl amine groups.
FIG. 11 is a representative synthetic scheme leading to exemplary
compounds of the invention with backbones of varying length.
FIG. 12 is a representative synthetic scheme leading to an
exemplary compound of the invention having a linker on the backbone
providing a locus for attaching the compound to another
molecule.
FIG. 13 is a normalized excitation (dotted line, .lambda..sub.an
=417 mn) and emission (full line, .lambda..sub.ex =350 nm) spectra
of the ligand H22IAM .about.10.sup.-6 M in Millipore water.
FIG. 14 is a UV/V is spectrum of [Tb(H22IAM)]+8.2.10.sup.-7 M in
Millipore water, 1.000 cm cell.
FIG. 15 is a normalized emission spectra of [Tb(H22IAM)]+ and
[Eu(H22IAM)]+ in millipore water. [Tb(H22IAM)]+ 8.2.10-7 M,
.lambda..sub.ex =354 nm; [Eu(H22IAM)]+ .about.10-6 M,
.lambda..sub.ex =350 nm.
FIG. 16 is an overlay plot of emission spectra of the complex
[Tb(H22IAM)].sup.+ in phosphate buffer 0.01 M at various
concentration. .lambda..sub.ex =347 mn.
FIG. 17 is an overlay plot of normalized emission spectra of
[Tb(H22IAM)].sup.+ at various concentrations. .lambda..sub.ex =335
nm (9.98.10.sup.-5 M) and .lambda..sub.ex =347 nm for all others
concentrations.
FIG. 18 is an overlay plot of normalized emission spectra of
[Tb(bicappedH22IAM)].sup.+ at various concentrations.
.lambda..sub.ex =365 nm (9.19.multidot.10.sup.-5 M) and
.lambda..sub.ex =351 nm for all other concentrations.
FIG. 19 is a table displaying exemplary compounds of the
invention.
FIG. 20 is a schematic diagram of an exemplary multiplex assay of
the invention.
FIG. 21 is a synthetic scheme leading to compounds of the invention
having backbones of variable length.
DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED
EMBODIMENTS
Abreviations
As used herein, "PL," refers to the phthalamidyl derived ligands of
the invention. "PL" encompasses the ligands of the invention in
both their free state and when they have complexed one or more
metal ions. Moreover, "PL" encompasses ligands that include one or
more phthamidyl groups in combination with one or more
salicylamidyl groups ("PSL").
Definitions
Unless defined otherwise, all technical and scientific terms used
herein generally have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used herein and the laboratory
procedures in molecular biology, organic chemistry and nucleic acid
chemistry and hybridization described below are those well known
and commonly employed in the art. Standard techniques are used for
nucleic acid and peptide synthesis. Generally, enzymatic reactions
and purification steps are performed according to the
manufacturer's specifications. The techniques and procedures are
generally performed according to conventional methods in the art
and various general references (see generally, Sambrook et al.
MOLECULAR MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
which is incorporated herein by reference), which are provided
throughout this document. The nomenclature used herein and the
laboratory procedures in analytical chemistry, and organic
synthetic described below are those known and employed in the art.
Standard techniques, or modifications thereof, are used for
chemical syntheses and chemical analyses.
"Analyte", as used herein, means any compound or molecule of
interest for which a diagnostic test is performed, such as a
biopolymer or a small molecular bioactive material. An analyte can
be, for example, a protein, peptide, carbohydrate, polysaccharide,
glycoprotein, hormone, receptor, antigen, antibody, virus,
substrate, metabolite, transition state analog, cofactor,
inhibitor, drug, dye, nutrient, growth factor, etc., without
limitation.
As used herein, "energy transfer" refers to the process by which
the fluorescence emission of a fluorescent group is altered by a
fluorescence-modifying group. If the fluorescence-modifying group
is a quenching group, then the fluorescence emission from the
fluorescent group is attenuated (quenched). Energy transfer can
occur through fluorescence resonance energy transfer, or through
direct energy transfer. The exact energy transfer mechanisms in
these two cases are different. It is to be understood that any
reference to energy transfer in the instant application encompasses
all of these mechanistically-distinct phenomena.
As used herein, "energy transfer pair" refers to any two molecules
that participate in energy transfer. Typically, one of the
molecules acts as a fluorescent group, and the other acts as a
fluorescence-modifying group. The preferred energy transfer pair of
the instant invention comprises a fluorescent group and a quenching
group of the invention. There is no limitation on the identity of
the individual members of the energy transfer pair in this
application. All that is required is that the spectroscopic
properties of the energy transfer pair as a whole change in some
measurable way if the distance between the individual members is
altered by some critical amount.
"Energy transfer pair" is used to refer to a group of molecules
that form a single complex within which energy transfer occurs.
Such complexes may comprise, for example, two fluorescent groups,
which may be different from one another and one quenching group,
two quenching groups and one fluorescent group, or multiple
fluorescent groups and multiple quenching groups. In cases where
there are multiple fluorescent groups and/or multiple quenching
groups, the individual groups may be different from one
another.
As used herein, "fluorescence-modifying group" refers to a molecule
of the invention that can alter in any way the fluorescence
emission from a fluorescent group. A fluorescence-modifying group
generally accomplishes this through an energy transfer mechanism.
Depending on the identity of the fluorescence-modifying group, the
fluorescence emission can undergo a number of alterations,
including, but not limited to, attenuation, complete quenching,
enhancement, a shift in wavelength, a shift in polarity, and a
change in fluorescence lifetime. One example of a
fluorescence-modifying group is a quenching group.
"Fluorescence resonance energy transfer" or "FRET" is used
interchangeably with FET, and refers to an energy transfer
phenomenon in which the light emitted by the excited fluorescent
group is absorbed at least partially by a fluorescence-modifying
group of the invention. If the fluorescence-modifying group is a
quenching group, then that group will preferably not radiate a
substantial fraction of the absorbed light as light of a different
wavelength, and will preferably dissipate it as heat. FRET depends
on an overlap between the emission spectrum of the fluorescent
group and the absorption spectrum of the quenching group. FRET also
depends on the distance between the quenching group and the
fluorescent group.
"Moiety" refers to the radical of a molecule that is attached to
another moiety.
As used herein, "nucleic acid" means DNA, RNA, single-stranded,
double-stranded, or more highly aggregated hybridization motifs,
and any chemical modifications thereof. Modifications include, but
are not limited to, those providing chemical groups that
incorporate additional charge, polarizability, hydrogen bonding,
electrostatic interaction, and fluxionality to the nucleic acid
ligand bases or to the nucleic acid ligand as a whole. Such
modifications include, but are not limited to, peptide nucleic
acids, phosphodiester group modifications (e.g., phosphorothioates,
methylphosphonates), 2'-position sugar modifications, 5-position
pyrimidine modifications, 8-position purine modifications,
modifications at exocyclic amines, substitution of 4-thiouridine,
substitution of 5-bromo or 5-iodo-uracil; backbone modifications,
methylations, unusual base-pairing combinations such as the
isobases, isocytidine and isoguanidine and the like. Modifications
can also include 3' and 5' modifications such as capping with a PL,
a fluorophore or another moiety.
As used herein, "quenching group" refers to any
fluorescence-modifying group of the invention that can attenuate at
least partly the light emitted by a fluorescent group. This
attenuation is referred to herein as "quenching". Hence,
illumination of the fluorescent group in the presence of the
quenching group leads to an emission signal that is less intense
than expected, or even completely absent. Quenching typically
occurs through energy transfer between the fluorescent group and
the quenching group.
"Peptide" refers to a polymer in which the monomers are amino acids
and are joined together through amide bonds, alternatively referred
to as a polypeptide. When the amino acids are .alpha.-amino acids,
either the L-optical isomer or the D-optical isomer can be used.
Additionally, unnatural amino acids, for example, .beta.-alanine,
phenylglycine and homoarginine are also included. Commonly
encountered amino acids that are not gene-encoded may also be used
in the present invention. All of the amino acids used in the
present invention may be either the D- or L-isomer. The L-isomers
are generally preferred. In addition, other peptidomimetics are
also useful in the present invention. For a general review, see,
Spatola, A. F., in Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New York,
p. 267 (1983).
The term "alkyl" is used herein to refer to a branched or
unbranched, saturated or unsaturated, monovalent hydrocarbon
radical, generally having from about 1-30 carbons and preferably,
from 4-20 carbons and more preferably from 6-18 carbons. When the
alkyl group has from 1-6 carbon atoms, it is referred to as a
"lower alkyl." Suitable alkyl radicals include, for example,
structures containing one or more methylene, methine and/or methyne
groups. Branched structures have a branching motif similar to
i-propyl, t-butyl, i-butyl, 2-ethylpropyl, etc. As used herein, the
term encompasses "substituted alkyls," and "cyclic alkyl."
"Substituted alkyl" refers to alkyl as just described including one
or more substituents such as lower alkyl, aryl, acyl, halogen
(i.e., alkylhalos, e.g., CF.sub.3), hydroxy, amino, alkoxy,
alkylamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl,
mercapto, thia, aza, oxo, both saturated and unsaturated cyclic
hydrocarbons, heterocycles and the like. These groups may be
attached to any carbon or substituent of the alkyl moiety.
Additionally, these groups may be pendent from, or integral to, the
alkyl chain.
The term "aryl" is used herein to refer to an aromatic substituent,
which may be a single aromatic ring or multiple aromatic rings
which are fused together, linked covalently, or linked to a common
group such as a methylene or ethylene moiety. The common linking
group may also be a carbonyl as in benzophenone. The aromatic
ring(s) may include phenyl, naphthyl, biphenyl, diphenylmethyl and
benzophenone among others. The term "aryl" encompasses "arylalkyl"
and "substituted aryl."
"Substituted aryl" refers to aryl as just described including one
or more functional groups such as lower alkyl, acyl, halogen,
alkylhalos (e.g. CF.sub.3), hydroxy, amino, alkoxy, alkylamino,
acylamino, acyloxy, phenoxy, mercapto and both saturated and
unsaturated cyclic hydrocarbons which are fused to the aromatic
ring(s), linked covalently or linked to a common group such as a
methylene or ethylene moiety. The linking group may also be a
carbonyl such as in cyclohexyl phenyl ketone. The term "substituted
aryl" encompasses "substituted arylalkyl."
The term "arylalkyl" is used herein to refer to a subset of "aryl"
in which the aryl group is attached to another group by an alkyl
group as defined herein.
"Substituted arylalkyl" defines a subset of "substituted aryl"
wherein the substituted aryl group is attached to another group by
an alkyl group as defined herein.
The term "acyl" is used to describe a ketone substituent, --(O)R,
where R is alkyl or substituted alkyl, aryl or substituted aryl as
defined herein.
The term "halogen" is used herein to refer to fluorine, bromine,
chlorine and iodine atoms.
The term "hydroxy" is used herein to refer to the group --OH.
The term "amino" is used to --NRR', wherein R and R' are
independently H, alkyl, aryl or substituted analogues thereof.
"Amino" encompasses "alkylamino" denoting secondary and tertiary
amines and "acylamino" describing the group RC(O)NR'.
The term "alkoxy" is used herein to refer to the --OR group, where
R is alkyl, or a substituted analogue thereof. Suitable alkoxy
radicals include, for example, methoxy, ethoxy, t-butoxy, etc.
As used herein, the term "aryloxy" denotes aromatic groups that are
linked to another group directly through an oxygen atom. This term
encompasses "substituted aryloxy" moieties in which the aromatic
group is substituted as described above for "substituted aryl."
Exemplary aryloxy moieties include phenoxy, substituted phenoxy,
benzyloxy, phenethyloxy, etc.
As used herein "aryloxyalkyl" defines aromatic groups attached,
through an oxygen atom to an alkyl group, as defined herein. The
term "aryloxyalkyl" encompasses "substituted aryloxyalkyl" moieties
in which the aromatic group is substituted as described for
"substituted aryl."
As used herein, the term "mercapto" defines moieties of the general
structure --S--R wherein R is H, alkyl, aryl or heterocyclic as
described herein.
The term "saturated cyclic hydrocarbon" denotes groups such as the
cyclopropyl, cyclobutyl, cyclopentyl, etc., and substituted
analogues of these structures. These cyclic hydrocarbons can be
single- or multi-ring structures.
The term "unsaturated cyclic hydrocarbon" is used to describe a
monovalent non-aromatic group with at least one double bond, such
as cyclopentene, cyclohexene, etc. and substituted analogues
thereof. These cyclic hydrocarbons can be single- or multi-ring
structures.
The term "heteroaryl" as used herein refers to aromatic rings in
which one or more carbon atoms of the aromatic ring(s) are replaced
by a heteroatom such as nitrogen, oxygen or sulfur. Heteroaryl
refers to structures that may be a single aromatic ring, multiple
aromatic ring(s), or one or more aromatic rings coupled to one or
more non-aromatic ring(s). In structures having multiple rings, the
rings can be fused together, linked covalently, or linked to a
common group such as a methylene or ethylene moiety. The common
linking group may also be a carbonyl as in phenyl pyridyl ketone.
As used herein, rings such as thiophene, pyridine, isoxazole,
phthalimide, pyrazole, indole, furan, etc. or benzo-fused analogues
of these rings are defined by the term "heteroaryl."
"Heteroarylalkyl" defines a subset of "heteroaryl" wherein an alkyl
group, as defined herein, links the heteroaryl group to another
group.
"Substituted heteroaryl" refers to heteroaryl as just described
wherein the heteroaryl nucleus is substituted with one or more
functional groups such as lower alkyl, acyl, halogen, alkylhalos
(e.g. CF.sub.3), hydroxy, amino, alkoxy, alkylamino, acylamino,
acyloxy, mercapto, etc. Thus, substituted analogues of
heteroaromatic rings such as thiophene, pyridine, isoxazole,
phthalimide, pyrazole, indole, furan, etc. or benzo-fused analogues
of these rings are defined by the term "substituted
heteroaryl."
"Substituted heteroarylalkyl" refers to a subset of "substituted
heteroaryl" as described above in which an alkyl group, as defined
herein, links the heteroaryl group to another group.
The term "heterocyclic" is used herein to describe a monovalent
saturated or unsaturated non-aromatic group having a single ring or
multiple condensed rings from 1-12 carbon atoms and from 1-4
heteroatoms selected from nitrogen, sulfur or oxygen within the
ring. Such heterocycles are, for example, tetrahydrofuran,
morpholine, piperidine, pyrrolidine, etc.
The term "substituted heterocyclic" as used herein describes a
subset of "heterocyclic" wherein the heterocycle nucleus is
substituted with one or more functional groups such as lower alkyl,
acyl, halogen, alkylhalos (e.g. CF.sub.3), hydroxy, amino, alkoxy,
alkylamino, acylamino, acyloxy, mercapto, etc.
The term "heterocyclicalkyl" defines a subset of "heterocyclic"
wherein an alkyl group, as defined herein, links the heterocyclic
group to another group.
Introduction
The present invention provides a class of luminescent probes that
are based on metal chelates of phthalamidyl-based ligands ("PL"),
particularly chelates of the lanthanide series. Other compounds of
the invention include both phthamidyl and salicylamidyl moieties in
a single ligand ("PSL"). The compounds of the invention emit light
or they can be used to absorb light emitted by a reporter
fluorophore. The fluorophores of the invention can be used as small
molecules in solution assays or they can be utilized as a label
that is attached to an analyte or a species that interacts with,
and allows detection and/or quantification of an analyte.
Fluorescent labels have the advantage of requiring few precautions
in their handling, and being amenable to high-throughput
visualization techniques (optical analysis including digitization
of the image for analysis in an integrated system comprising a
computer). Preferred labels are typically characterized by one or
more of the following: high sensitivity, high stability, low
background, long lifetimes, low environmental sensitivity and high
specificity in labeling.
The fluorophores of the invention can be used with other
fluorophores or quenchers as components of energy transfer probes.
Many fluorescent labels are useful in combination with the PL and
PSL of the invention. Many such labels are commercially available
from, for example, the SIGMA chemical company (Saint Louis, Mo.),
Molecular Probes (Eugene, Oreg.), R&D systems (Minneapolis,
Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH
Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich
Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL
Life Technologies, Inc. (Gaithersburg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
and Applied Biosystems (Foster City, Calif.), as well as many other
commercial sources known to one of skill. Furthermore, those of
skill in the art will recognize how to select an appropriate
fluorophore for a particular application and, if it not readily
available conmmercially, will be able to synthesize the necessary
fluorophore de novo or synthetically modify commercially available
fluorescent compounds to arrive at the desired fluorescent
label.
In addition to small molecule fluorophores, naturally occurring
fluorescent proteins and engineered analogues of such proteins are
useful with the PLs and PSLs of the present invention. Such
proteins include, for example, green fluorescent proteins of
cnidarians (Ward et al., Photochem. Photobiol. 35:803-808 (1982);
Levine et al., Comp. Biochem. Physiol., 72B:77-85 (1982)), yellow
fluorescent protein from Vibrio fischeri strain (Baldwin et al.,
Biochemistry 29:5509-15 (1990)), Peridinin-chlorophyll from the
dinoflagellate Symbiodinium sp. (Morris et al., Plant Molecular
Biology 24:673:77 (1994)), phycobiliproteins from marine
cyanobacteria, such as Synechococcus, e.g., phycoerythrin and
phycocyanin (Wilbanks et al., J. Biol. Chem. 268:1226-35 (1993)),
and the like.
The compounds of the invention can be used as probes, as tools for
separating particular ions from other solutes, as probes in
microscopy, enzymology, clinical chemistry, molecular biology and
medicine. The compounds of the invention are also useful as
therapeutic agents in modalities, such as photodynamic therapy and
as diagnostic agents in imaging methods, such as magnetic resonance
imaging. Moreover, the compounds of the invention are useful as
components of optical amplifiers of light, waveguides and the like.
Furthermore, the compounds of the invention can be incorporated
into inks and dyes, such as those used in the printing of currency
or other negotiable instruments.
The compounds of the invention can be made to luminesce by exciting
them in any manner known in the art, including, for example, with
light or electrochemical energy (see, for example, Kulmala et al,
Analytica Chimica Acta 386: 1 (1999)). The luminescence can, in the
case of chiral compounds of the invention, be circularly polarized
(see, for example, Riehl et al., Chem. Rev. 86: 1 (1986)).
The compounds, probes and methods discussed in the following
sections are generally representative of the compositions of the
invention and the methods in which such compositions can be used.
The following discussion is intended as illustrative of selected
aspects and embodiments of the present invention and it should not
be interpreted as limiting the scope of the present invention.
The Compounds
The present invention provides an anay of phthalamidyl-based metal
chelating ligands ("PL") that comprise at least one phthamidyl
moiety within their framework. The PL compounds can also include
one or more salicylamidyl moiety within their framework in
combination with the one or more phthalamidyl moiety.
In one aspect, the invention provides a luminescent lanthanide ion
complex. The chelating group comprises at least one phthalamidyl
group, preferably between 2 and 100 phthalamidyl groups, more
preferably between 3 and 75 phthamidyl groups, even more preferably
between 4 and 50 phthalamidyl groups and more preferably still,
between 5 and 25 phthamidyl groups. The complex also, preferably
has a quantum yield of at least about 0.1. Even more preferably,
the lanthanide ion of the complex is a member selected from
europium, terbium and combinations thereof.
The at least one phthalamidyl group of the chelating group can be
substituted with one or more electron withdrawing and/or electron
donating group. Those of skill in the art will understand which
substituents, when appended to an aromatic ring will exhibit
electron withdrawing or electron donating properties. Tables of
substituents that are appropriate for inclusion in the PLs of the
invention can be found in the literature. See, for example,
Hammett, J. Am. Chem. Soc. 59: 96 (1937); Johnson, THE HAMMET
EQUATION, Cambridge University Press, New York, 1973; Hansch et
al., J. Med. Chem. 16: 1207 (1973); and Hansch et al., SUBSTITUENT
CONSTANTS FOR CORRELATION ANALAYSIS IN CHEMISTRY AND BIOLOGY,
Wiley, New York, 1979.
Moreover, the phthalamidyl groups of the complex can be connected
by a backbone of substantially any length and chemical composition,
with the proviso that the backbone should orient the phthalamidyl
and other rings in a manner that is conducive to their complexation
of the desired metal ion. That the backbone be stable to the
conditions in which the complex is used is also generally
preferred. As such, representative backbones include, for example,
alkyl groups, substituted alkyl groups, conjugated unsaturated
systems, aryl groups, heteroaryl groups, dendrimers, polyethers,
polyamides, polyimines, biopolymers and backbones that are a
combination of more than one of these groups. Other useful backbone
systems will be apparent to those of skill in the art.
In a second aspect, the present invention provides a compound
having a structure according to Formula I: ##STR2##
In Formula I, the groups R.sup.1, R.sup.2, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.10 and R.sup.20 are members independently
selected from the group consisting of H, alkyl and substituted
alkyl groups, wherein, two or more of R.sup.2, R.sup.4, R.sup.5,
R.sup.7 and, when R.sup.3 is substituted alkyl, a substituent of
R.sup.3 are optionally adjoined by at least one linker moiety to
form at least one ring. R.sup.3, R.sup.8 and R.sup.9 are members
independently selected from the group consisting of alkyl,
substituted alkyl, aryl and substituted aryl groups. R.sup.11,
R.sup.12, R.sup.13, R.sup.21, R.sup.22 and R.sup.23 are members
independently selected from alkyl, substituted alkyl, H,
--NR.sup.14 R.sup.15, --NO.sub.2, --OR.sup.16, --COOR.sup.17,
wherein, R.sup.14, R.sup.15, R.sup.16 and R.sup.17 are members
independently selected from the group consisting of H, alkyl and
substituted alkyl, wherein R.sup.12 can optionally form a ring with
R.sup.11, R.sup.13 or both, and R.sup.22 can optionally form a ring
with R.sup.21, R.sup.23 or both. The rings are members
independently selected from the group of ring systems consisting of
cyclic alkyl, substituted cyclic alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclyl and saturated
heterocyclyl ring systems. Q.sup.1 is --OR.sup.18 and Q.sup.2 is
--OR.sup.9. R.sup.18 and R.sup.19 are members independently
selected from H, an enzymatically labile group, a hydrolytically
labile group and a single negative charge. The number represented
by a is 0 or 1, with the proviso that when a is 0, N.sup.2 is
covalently attached directly to carbonyl group 2'. Similarly, z is
0 or 1, with the proviso that when z is 0, N.sup.1' is covalently
attached directly to carbonyl group 1'.
Although the complexing agents of the invention can have any useful
number of phthalamidyl rings, in a preferred embodiment, z is 0,
generally affording a complexing agent having three phthalamidyl
rings.
As discussed above, the backbone, here represented by R.sup.3, can
have any useful structure and size, so long as it is functionalized
to allow at least one phthamidyl group to be attached thereto. In a
preferred embodiment, R.sup.3 is a linear C.sub.1 -C.sub.6
hydrocarbon.
In another preferred embodiment, the present invention provides a
compound having three phthalamidyl rings. The compound has a
structure according to Formula I, in which R.sup.8 is
(CH.sub.2).sub.P, wherein P is selected from the group consisting
of the integers from 1 to 5, inclusive. R.sup.4 provides the third
phthalamidyl ring and is an alkyl group substituted with a moiety
having a structure according to Formula II: ##STR3##
In Formula II, R.sup.29, R.sup.46 and R.sup.47 are members
independently selected from the group consisting of H, alkyl and
substituted alkyl groups, wherein, two or more of R.sup.2, R.sup.7
and R.sup.29 are optionally adjoined by at least one linker moiety
to form at least one ring. R.sup.31, R.sup.32 and R.sup.33 are
members independently selected from alkyl, substituted alkyl, H,
--NR.sup.24 R.sup.25, --NO.sub.2, --OR.sup.26, --COOR.sup.27,
wherein, R.sup.24, R.sup.25, R.sup.26 and R.sup.27 are members
independently selected from the group consisting of H, alkyl and
substituted alkyl, wherein R.sup.32 can optionally form a ring with
R.sup.31, R.sup.33 or both. The rings are members independently
selected from the group of ring systems consisting of cyclic alkyl,
substituted cyclic alkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, heterocyclyl and saturated heterocyclyl
ring systems. R.sup.3 is (CH.sub.2).sub.X, wherein P is selected
from the group consisting of the integers from 1 to 5, inclusive.
Q.sup.3 --OR.sup.28, wherein R.sup.28 is a member selected from H,
an enzymatically labile group, a hydrolytically labile group and a
single negative charge. In this embodiment, z from Formula I is
0.
In further preferred embodiment, the compounds of the invention are
macrocyclic or polymacrocyclic structures. Representative of this
embodiment is a compound whose structure includes the moieties set
forth in Formulae I and II and in which two or more of R.sup.2,
R.sup.7 and R.sup.29 are adjoined by at least one linker moiety to
form at least one ring, more preferably, R.sup.2, R.sup.7 and
R.sup.29 together comprise a single linker moiety.
Although the linker moieties used to form the macrocyclic or
polymacrocyclic structures can have substantially any useful
structure, including for example, alkyl, substituted alkyl,
arylalkyl, heteroarylalkyl, conjugated unsaturated systems, aryl
groups, heteroaryl groups, dendrimers, polyethers, polyamides,
polyimines, biopolymers and backbones that are a combination of
more than one of these groups, a presently preferred linker has a
structure according to Formula III: ##STR4##
In Formula III, b, e and f are members independently selected from
the group consisting of the integers from 1 to 5, inclusive.
In a still further preferred embodiment, the invention provides a
compound having a structure according to Formula IV: ##STR5##
In Formula IV, b, b', e, e', f and f' are members independently
selected from the group consisting of the integers from 1 to 5,
inclusive.
In still further preferred embodiments, the invention provides
compounds having a structure according to Formula V: ##STR6##
or more preferably still, Formula VI: ##STR7##
In Formulae V and VI, R.sup.1, R.sup.2, R.sup.10 and Q.sup.1
represent the same species as those described in conjunction with
the compound according to Formula I. In each of Formulae IV-VI, the
substituents (R) are substantially as described above.
In another preferred embodiment, the invention provides a compound
with a minimum of four phthalamidyl rings. The compound has a
structure according to Formula I, wherein R.sup.4 is an alkyl group
substituted with a group having a structure according to Formula
II, above. The fourth phthalamidyl ring is provided by R.sup.5,
which is an alkyl group substituted with a moiety having a
structure according to Formula IX: ##STR8##
In Formula IX, R.sup.39, R.sup.40 and R.sup.45 are members
independently selected from alkyl and substituted alkyl groups.
R.sup.41, R.sup.42 and R.sup.43 are members independently selected
from alkyl, substituted alkyl, H, --NR.sup.34 R.sup.35, --NO.sub.2,
--OR.sup.36, --COOR.sup.37, wherein, R.sup.34, R.sup.35, R.sup.36
and R.sup.37 are members independently selected from the group
consisting of H, alkyl and substituted alkyl, wherein R.sup.42 can
optionally form a ring with R.sup.41, R.sup.43 or both. The rings
are members independently selected from the group of ring systems
consisting of cyclic alkyl, substituted cyclic alkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl
and saturated heterocyclyl ring systems.
In another preferred embodiment, the invention provides a compound
having a structure according to Formula X: ##STR9##
In Formula X, the letters M, N, P and Z represent numbers that are
members independently selected from the group consisting of the
integers between 1 and 5, inclusive.
In another preferred embodiment, the present invention provides a
having a structure according to Formula XI: ##STR10##
The substituents (R) in Formula XI, have substantially the same
identity as identically named substituents in the Formulae
above.
In a further preferred example, the invention provides a compound
combining Formulae I, II and IX, and in which R.sup.1, R.sup.6,
R.sup.29 and R.sup.39 together comprise a single linker moiety. The
linker preferably has a structure according to Formula XII:
##STR11##
In Formula XII, b, e, f, g and h are members independently selected
from the numbers between 1 and 5, inclusive.
In a still further preferred embodiment, the invention provides a
compound having a structure according to Formula XIII:
##STR12##
In Figure XIII, b and b' are members independently selected from
the group consisting of the integers from 1 to 5, inclusive; e, e',
f, f', g, g', h and h' are members independently selected from the
group consisting of numbers from 0 to 3. IN each of Formulae
X-XIII, the substituents (R") are substantially as described
above.
In a still further preferred embodiment, the invention provides
compounds having a structure according to Formula XIV:
##STR13##
In another preferred embodiment, the backbone of the compound is a
dendrimer. Thus, the invention provides compounds having a
structure according to Formula XV: ##STR14##
In Formula XV, D represents the dendrimer; and w is a member
selected from the group consisting of the integers from 4 to 100,
inclusive, more preferably between 8 and 50, inclusive.
The PL of the invention can also form macrocyclic ligands, which
are bound to the dendrimer or which incorporate constituents of the
dendrimer into the macrocyclic framework. A representative of such
compounds have has a structure according to Formula XVI:
##STR15##
in which D and w are as set forth above. It will be apparent to one
of skill that the dendrimer and the ligand in each of these
structures can be linked by any type of spacer arm, including, for
example an amine, such as the backbone amines of the present
invention.
In a preferred embodiment of the compounds of Formulae XV and XVI,
the dendrimer is a poly(propyleneimine) dendrimer, preferably of
generation 2 to generation 10, inclusive.
In yet another preferred embodiment, the invention provides
polymacrocyclic compounds. A representative structure of a compound
of this embodiment of the invention is set forth in Formula XVII:
##STR16##
R Groups
For clarity of illustration, the discussion of the identities of
the various R groups (e.g., R.sup.1, R.sup.2, R.sup.3, etc.) set
forth in the Formulae above is collected together in this section.
This discussion is equally applicable to each of the formulae set
forth herein. Moreover, although the discussion focuses on certain
representative formulae, it is to be understood that this is a
device used to simplify the discussion of the R groups and that it
does not serve to limit the scope of the R groups.
Referring to Formulae I and II in combination and the resulting
complexing agent with three phthalamidyl rings, the following
discussion is generally relevant to any compound of the invention
and any R group of any compound of the invention. Moreover, this
discussion is specifically relevant to the R groups R.sup.1,
R.sup.2, R.sup.3, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.29, R.sup.46 and R.sup.47. It will be apparent to
those of skill in the art that when additional phthalanmdyl rings,
linker groups and backbones are included in a compound of the
invention, the following discussion is equally relevant to
them.
In one preferred embodiment, one or more of the above-recited R
groups is a member independently selected from the group consisting
of H, C.sub.1 to C.sub.10 alkyl and C.sub.1 to C.sub.10 substituted
alkyl, and more preferably members independently selected from the
group consisting of H, C.sub.2 to C.sub.6 alkyl and C.sub.2 to
C.sub.6 substituted alkyl.
In another preferred embodiment, one or more of the above-recited R
groups is a member independently selected from the group consisting
of H, aryl, substituted aryl and combinations thereof.
In a further preferred embodiment, one or more of the above-recited
R groups is a member independently selected from the group
consisting of H and alkyl substituted with polycyclic aryl groups,
preferably napthyl groups.
In yet another preferred embodiment, one or more of the
above-recited R groups is a member selected from the group
consisting of H and a primary alkyl amine, preferably a C.sub.1 to
C.sub.10 alkyl chain bearing an amine moiety at the
.omega.-position, more preferably a C.sub.2 to C.sub.6 alkyl chain
bearing an amine moiety at the .omega.-position.
In a still further preferred embodiment, one or more of the
above-recited R groups is a polyether, preferably a member selected
from ethylene glycol, ethylene glycol oligomers and combinations
thereof, having a molecular weight of from about 60 daltons to
about 10,000 daltons, and more preferably of from about 100 daltons
to about 1,000 daltons.
Representative polyether-based substituents include, but are not
limited to, the following structures: ##STR17##
in which j is a number from 1 to 100, inclusive. Other
functionalized polyethers are known to those of skill in the art,
and many are commercially available from, for example, Shearwater
Polymers, Inc. (Alabama).
In another preferred embodiment, one or more of the above-recited R
groups comprise a reactive group for conjugating said compound to a
member selected from the group consisting of molecules and
surfaces. Representative useful reactive groups are discussed in
greater detail in the succeeding section. Additional information on
useful reactive groups is known to those of skill in the art. See,
for example, Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press,
San Diego, 1996.
In a preferred embodiment, one or more of the above-recited R
groups is a member selected from .omega.-carboxyl alkyl groups,
.omega.-carboxyl substituted alkyl groups and combinations thereof,
more preferably the R group has a structure according to Formula
VII: ##STR18##
In Formula VII, X is a member selected from O, S and NR.sup.50.
R.sup.50 is a member selected from H, alkyl and substituted alkyl.
Y is a member selected from H and a single negative charge; and j
and k are members independently selected from the group consisting
of integers from 1 to 18.
In a further preferred embodiment, one or more of the above-recited
R groups has a structure according to Formula VIII: ##STR19##
in which Y is substantially as recited above for Formula VII.
In yet another preferred embodiment, one or more of the R groups
can combine characteristics of one or more of the above-recited
groups. For example, one preferred R group combines both the
attributes of a polyether and a reactive group: ##STR20##
in which j is an integer between 1 and 100, inclusive. Other
"chimeric" R groups include, but are not limited to, moieties such
as sugars (e.g., polyol with reactive hydroxyl), amino acids, amino
alcohols, carboxy alcohols, amino thiols, and the like.
In a still further preferred embodiment, the compounds of the
invention have more than one type of R group on a single molecule.
For example a single molecule can include an R group that is a
polyether and an R group that is an amine. Many other such
combinations of different substituents will be apparent to those of
skill in the art. Representative structures according to this
embodiment are set forth below: ##STR21##
wherein, n is an integer between 0 and 6, and preferably between 1
and 3.
Structural illustrations of certain exemplary compounds of the
invention are set forth in FIG. 19.
Exemplary lanthanide chelates of the invention have a structure
according to Structure 1: ##STR22##
where s is between 1 and 5, inclusive. Exemplary preferred
compounds are set forth in Table 1.
TABLE 1 Denticity Structure R.sup.0 R"" 4 acyclic 3(M)Li
methylamine 4 macrocyclic 5Li 5Li 6 acyclic TREN methylamine 6
macrobicyclic TREN 2-AMN 8 acyclic H22 methylamine 8 macrotricyclic
H22 H22 For all compounds in table 1, R', R", and R"' are H and
R.sup.0 and R"" are amides; 3(M)Li =
2,2-dimethyl-1,3-diaminopropane; 5LI = 1,5-diaminopentane; TREN =
tris(2-aminoethyl)amine; 2-AMN = 2-aminomethylnaphthalene; H22 =
tetrakis(2-aminoethyl)ethylenediamine
In yet another preferred embodiment, the compounds of the invention
are associated with another molecule by a weak interaction (e.g.
van der Waals) to form a species, such as, for example, and
inclusion complex. Preferred molecules interacting with the PLs
include, but are not limited to dendrimers, macrocycles,
cyclodextrins, and the like.
Reactive Functional Groups
Certain of the compounds of the invention bear a reactive
functional group, such as a component of a linker arm, which can be
located at any position on any aryl nucleus or on a chain, such as
an alkyl chain, attached to an aryl nucleus, or on the backbone of
the chelating agent. These compounds are referred to herein as
"reactive ligands." When the reactive group is attached to an
alkyl, or substituted alkyl chain tethered to an aryl nucleus, the
reactive group is preferably located at a terminal position of an
alkyl chain. Reactive groups and classes of reactions useful in
practicing the present invention are generally those that are well
known in the art of bioconjugate chemistry. Currently favored
classes of reactions available with reactive ligands of the
invention are those which proceed under relatively mild conditions.
These include, but are not limited to nucleophilic substitutions
(e.g., reactions of amines and alcohols with acyl halides, active
esters), electrophilic substitutions (e.g. enamine reactions) and
additions to carbon-carbon and carbon-heteroatom multiple bonds
(e.g., Michael reaction, Diels-Alder addition). These and other
useful reactions are discussed in, for example, March, ADVANCED
ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985;
Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,
1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in
Chemistry Series, Vol. 198, American Chemical Society, Washington,
D.C., 1982.
Useful reactive functional groups include, for example: (a)
carboxyl groups and various derivatives thereof including, but not
limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole
esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl
esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl
groups, which can be converted to esters, ethers, aldehydes, etc.
(c) haloalkyl groups, wherein the halide can be later displaced
with a nucleophilic group such as, for example, an amine, a
carboxylate anion, thiol anion, carbanion, or an alkoxide ion,
thereby resulting in the covalent attachment of a new group at the
site of the halogen atom; (d) dienophile groups, which are capable
of participating in Diels-Alder reactions such as, for example,
maleimido groups; (e) aldehyde or ketone groups, such that
subsequent derivatization is possible via formation of carbonyl
derivatives such as, for example, imines, hydrazones,
semicarbazones or oximes, or via such mechanisms as Grignard
addition or alkyllithium addition; (f) sulfonyl halide groups for
subsequent reaction with amines, for example, to form sulfonamides;
(g) thiol groups, which can be converted to disulfides or reacted
with acyl halides; (h) amine or sulfhydryl groups, which can be,
for example, acylated, alkylated or oxidized; (i) alkenes, which
can undergo, for example, cycloadditions, acylation, Michael
addition, etc; (j) epoxides, which can react with, for example,
amines and hydroxyl compounds; and (k) phosphoramidites and other
standard functional groups useful in nucleic acid synthesis.
The reactive functional groups can be chosen such that they do not
participate in, or interfere with, the reactions necessary to
assemble the reactive ligand. Alternatively, a reactive functional
group can be protected from participating in the reaction by the
presence of a protecting group. Those of skill in the art
understand how to protect a particular functional group such that
it does not interfere with a chosen set of reaction conditions. For
examples of useful protecting groups, see, for example, Greene et
al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons,
New York, 1991.
Donor and Acceptor Moieties
One of the advantages of the compounds of the invention is that
they can be used with a wide range of energy donor and acceptor
molecules to construct fluorescence energy transfer probes. A vast
array of fluorophores useful in conjunction with the PLs are known
to those of skill in the art. See, for example, Cardullo et al.,
Proc. Natl. Acad. Sci. USA 85: 8790-8794 (1988); Dexter, D. L., J.
of Chemical Physics 21: 836-850 (1953); Hochstrasser et al.,
Biophysical Chemistry 45: 133-141 (1992); Selvin, P., Methods in
Enzymology 246: 300-334 (1995); Steinberg, I. Ann. Rev. Biochem.,
40: 83-114 (1971); Stryer, L. Ann. Rev. Biochem., 47: 819-846
(1978); Wang et al., Tetrahedron Letters 31: 6493-6496 (1990); Wang
et al., Anal. Chem. 67: 1197-1203 (1995).
A non-limiting list of exemplary donors that can be used in
conjunction with the quenchers of the invention is provided in
Table 2.
TABLE 2 Suitable moieties that can be selected as donors or
acceptors in FET pairs
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid acridine
and derivatives: acridine acridine isothiocyanate
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)
4-amio-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate
N-(4-anilino-1-naphthyl)maleimide anthranilamide BODIPY Brilliant
Yellow coumarin and derivatives: coumarin 7-amino-4-methylcoumarin
(AMC, Coumarin 120) 7-amino-4-trifluoromethylcouluarin (Coumaran
151) cyanine dyes cyanosine 4',6-diaminidino-2-phenylindole (DAPI)
5',5"-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin
diethylenetriamine pentaacetate
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid
4,4'-diisothiocyanatostilbene-2,2'-dissulfonic acid
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,
dansylchloride) 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL)
4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC) eosin and
derivatives: eosin eosin isothiocyanate erythrosin and derivatives:
erythrosin B erythrosin isothiocyanate ethidium fluorescein and
derivatives: 5-carboxyfluorescein (FAM)
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE)
fluorescein fluorescein isothiocyanate QFITC (XRITC) fluorescamine
IR144 IR1446 Malachite Green isothiocyanate 4-methylumbelliferone
ortho cresolphthalein nitrotyrosine pararosaniline Phenol Red
B-phycoerythrin o-phthaldialdehyde pyrene and derivatives: pyrene
pyrene butyrate succinimidyl 1-pyrene butyrate quantum dots
Reactive Red 4 (Cibacron .TM. Brilliant Red 3B-A) rhodamine and
derivatives: 6-carboxy-X-rhodamine (ROX) 6-carboxyrhodamine (R6G)
lissamine rhodamine B sulfonyl chloride rhodamine (Rhod) rhodamine
B rhodamine 123 rhodamine X isothiocyanate sulforhodamine B
sulforhodamine 101 sulfonyl chloride derivative of sulforhodamine
101 (Texas Red) N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA)
tetramethyl rhodamine tetramethyl rhodamine isothiocyanate (TRITC)
riboflavin rosolic acid lanthanide chelate derivatives
There is a great deal of practical guidance available in the
literature for selecting appropriate donor-acceptor pairs for
particular probes, as exemplified by the following references:
Pesce et al., Eds., FLUORESCENCE SPECTOROSCOPY (Marcel Dekker, New
York, 1971); White et al., FLUORESCENCE ANALYSIS: A Practical
Approach (Marcel Dekker, New York, 1970); and the like. The
literature also includes references providing exhaustive lists of
fluorescent and chromogenic molecules and their relevant optical
properties, for choosing reporter-quencher pairs (see, for example,
Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES,
2nd Edition (Academic Press, New York, 1971); Griffiths, COLOUR AND
CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976);
Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972); Haugland,
HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular
Probes, Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE
(Interscience Publishers, New York, 1949); and the like. Further,
there is extensive guidance in the literature for derivatizing
reporter and quencher molecules for covalent attachment via readily
available reactive groups that can be added to a molecule.
The diversity and utility of chemistries available for conjugating
fluorophores to other molecules and surfaces is exemplified by the
extensive body of litereature on preparing nucleic acids
derivatized with fluorophores. See, for example, Haugland (supra);
Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al., U.S. Pat.
No. 4,351,760. Thus, it is well within the abilities of those of
skill in the art to choose an energy exchange pair for a particular
application and to conjugate the members of this pair to a probe
molecule, such as, for example, a small molecular bioactive
material, nucleic acid, peptide or other polymer.
In a FET pair, it is generally preferred that an absorbance band of
the acceptor substantially overlap a fluorescence emission band of
the donor. When the donor (fluorophore) is a component of a probe
that utilizes fluorescence resonance energy transfer (FRET), the
donor fluorescent moiety and the quencher (acceptor) of the
invention are preferably selected so that the donor and acceptor
moieties exhibit fluorescence resonance energy transfer when the
donor moiety is excited. One factor to be considered in choosing
the fluorophore-quencher pair is the efficiency of fluorescence
resonance energy transfer between them. Preferably, the efficiency
of FRET between the donor and acceptor moieties is at least 10%,
more preferably at least 50% and even more preferably at least 80%.
The efficiency of FRET can easily be empirically tested using the
methods both described herein and known in the art.
The efficiency of FRET between the donor-acceptor pair can also be
adjusted by changing ability of the donor and acceptor to dimerize
or closely associate. If the donor and acceptor moieties are known
or determined to closely associate, an increase or decrease in
association can be promoted by adjusting the length of a linker
moiety, or of the probe itself, between the two fluorescent
proteins. The ability of donor-acceptor pair to associate can be
increased or decreased by tuning the hydrophobic or ionic
interactions, or the steric repulsions in the probe construct.
Thus, intramolecular interactions responsible for the association
of the donor-acceptor pair can be enhanced or attenuated. Thus, for
example, the association between the donor-acceptor pair can be
increased by, for example, utilizing a donor bearing an overall
negative charge and an acceptor with an overall positive
charge.
In addition to fluorophores that are attached directly to a probe,
the fluorophores can also be attached by indirect means. In this
embodiment, a ligand molecule (e.g., biotin) is preferably
covalently bound to the probe species. The ligand then binds to
another molecules (e.g., streptavidin) molecule, which is either
inherently detectable or covalently bound to a signal system, such
as a fluorescent compound of the invention, or an enzyme that
produces a fluorescent compound by conversion of a non-fluorescent
compound. Useful enzymes of interest as labels include, for
example, hydrolases, particularly phosphatases, esterases and
glycosidases, or oxidotases, particularly peroxidases. Fluorescent
compounds include fluorescein and its derivatives, rhodamine and
its derivatives, dansyl, umbelliferone, etc., as discussed above.
For a review of various labeling or signal producing systems that
can be used, see, U.S. Pat. No. 4,391,904.
Presently preferred fluorophores of use in conjunction with the
complexes of the invention, include, for example, xanthene dyes,
including fluoresceins, and rhodamine dyes. Many suitable forms of
these compounds are widely available commercially with substituents
on their phenyl moieties, which can be used as the site for bonding
or as the bonding functionality for attachment to an nucleic acid.
Another group of preferred fluorescent compounds are the
naphthylamines, having an amino group in the alpha or beta
position. Included among such napbthylamino compounds are
1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene
sulfonate and 2-p-touidinyl-6-naphthalene sulfonate. Other donors
include 3-phenyl-7-isocyanatocoumarin, acridines, such as
9-isothiocyanatoacridine and acridine orange;
N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes,
pyrenes, and the like.
For clarity of illustration, the discussion below focuses on
attaching the complexes of the invention and other fluorophores to
nucleic acids. The focus on nucleic acid probes is not intended to
limit the scope of probe molecules to which the complexes of the
invention can be attached. Those of skill in the art will
appreciate that the complexes of the invention can also be attached
to small molecules (e.g., small molecular bioactive agents),
proteins, peptides, synthetic polymers, solid supports and the like
using standard synthetic chemistry or modifications thereof.
In an exemplary embodiment, in which the probe is a nucleic acid
probe, the acceptor molecule is a rhodamine dye. The rhodamine
moiety is preferably attached to either the 3'- or the 5'-terminus
of the nucleic acid, although internal sites are also accessible
for derivitization of PLs and have utility for selected purposes.
Whichever terminus the rhodamine derivative is attached to, the
complex of the invention will generally be attached to its
antipode, or at a position internal to the nucleic acid chain. The
rhodamine acceptor is preferably introduced using a commercially
available amidite. Different donor groups of the invention are also
preferably introduced using a reactive derivative (e.g., amidite)
of the donor. Alternatively, donor groups comprising reactive
groups (e.g., isothiocyanates, active esters, etc.) can be
introduced via reaction with a reactive moiety on a tether or
linker arm attached to the nucleic acid (e.g., hexylamine).
In yet another preferred embodiment, the donor moiety can be
attached at the 3'-terminus of a nucleic acid by the use of a
derivatized synthesis support. For example, a complexing agent of
the invention is tethered to a solid support that is derivatized
with an analogue of the complex. Such derivatized supports are well
known in the art and are exemplified by a TAMRA
(tetramethylrhodamine carboxylic acid) derivative that is attached
to a nucleic acid 3'-terminus using a commercially available solid
support that is derivatized with an analogue of the TAMRA
fluorophore (Biosearch Technologies, Inc.)
In view of the well-developed body of literature concerning the
conjugation of small molecules to nucleic acids, many other methods
of attaching donor/acceptor pairs to nucleic acids will be apparent
to those of skill in the art. For example, rhodamine and
fluorescein dyes are conveniently attached to the 5'-hydroxyl of an
nucleic acid at the conclusion of solid phase synthesis by way of
dyes derivatized with a phosphoramidite moiety (see, for example,
Woo et al., U.S. Pat. No. 5,231,191; and Hobbs, Jr., U.S. Pat. No.
4,997,928).
More specifically, there are many linking moieties and
methodologies for attaching groups to the 5'- or 3'-termini of
nucleic acids, as exemplified by the following references:
Eckstein, editor, Nucleic Acids and Analogues: A Practical Approach
(IRL Press, Oxford, 1991); Zuckerman et al., Nucleic Acids
Research, 15: 5305-5321 (1987) (3'-thiol group on nucleic acid);
Sharma et al., Nucleic Acids Research, 19: 3019 (1991)
(3'-sulfhydryl); Giusti et al., PCR Methods and Applications, 2:
223-227 (1993) and Fung et al., U.S. Pat. No. 4,757,141
(5'-pbosphoamino group via Aminolink TM II available group);
Agrawal et al., Tetrahedron Letters, 31: 1543-1546 (1990)
(attachment via phosphoramidate linkages); Sproat et al., Nucleic
Acids Research, 15: 4837 (1987) (5-mercapto group); Nelson et al.,
Nucleic Acids Research, 17: 7187-7194 (1989) (3'-amino group), and
the like.
Means of detecting fluorescent labels are well known to those of
skill in the art. Thus, for example, fluorescent labels can be
detected by exciting the fluorophore with the appropriate
wavelength of light and detecting the resulting fluorescence. The
fluorescence can be detected visually, by means of photographic
film, by the use of electronic detectors such as charge coupled
devices (CCDs) or photomultipliers and the like. Similarly,
enzymatic labels may be detected by providing the appropriate
substrates for the enzyme and detecting the resulting reaction
product.
Synthesis
The compounds of the invention are synthesized by an appropriate
combination of generally well-known synthetic methods. Techniques
useful in synthesizing the compounds of the invention are both
readily apparent and accessible to those of skill in the relevant
art. The discussion below is offered to illustrate certain of the
diverse methods available for use in assembling the compounds of
the invention, it is not intended to limit the scope of reactions
or reaction sequences that are useful in preparing the compounds of
the present invention.
The compounds of the invention can be prepared as a single
stereoisomer or as a mixture of stereoisomers. In a preferred
embodiment, the compounds are prepared as substantially a single
isomer. Isomerically pure compounds are prepared by using synthetic
intermediates that are isomerically pure in combination with
reactions that either leave the stereochemistry at a chiral center
unchanged or result in its complete inversion. Alternatively, the
final product or intermediates along the synthetic route can be
resolved into a single stereoisomer. Techniques for inverting or
leaving unchanged a particular stereocenter, and those for
resolving mixtures of stereoisomers are well known in the art and
it is well within the ability of one of skill in the art to choose
an appropriate method for a particular situation. See, generally,
Furniss et al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC
CHEMISTRY 5.sup.th ED., Longman Scientific and Technical Ltd.,
Essex, 1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128
(1990).
An exemplary synthetic scheme leading to a bi-capped complexing
agent of the invention is set forth in Scheme 1 (FIG. 1). An
isophthalic acid derivative is activated at both carboxyl positions
by contacting the acid with an activating group, such as
2-mercaptothiazoline under dehydrating conditions to produce
compound 1. The activated carboxylic acid derivative 1 is tethered
to a backbone, such as a polyamine by reacting an excess of 1 with
the backbone component to produce compound 2. The ligand 2 is
converted into a macrocyclic analogue 3 by reaction with another
backbone (e.g., linker) molecule, which may be the same as or
different than the backbone utilized to form 2.
Another exemplary synthetic route is set forth in Scheme 2 (FIG.
8). 2-Methoxyisophthaloyl thiazoline 1, is reacted with a polyamine
ligand, such as tris(2-aminoethyl)amine ("TRENSAM") under
conditions of high dilution to acylate each of the amines of the
ligand, thereby forming compound 7. Compound 7 is susbsequently
reacted with an amine, such as methyl amine to form ligand 8.
A further exemplary synthetic scheme is set forth in Scheme 3 (FIG.
9). Compound 7 (Scheme 2) is treated with a linker arm including
both a reactive amine and a carboxylic acid group 16 to form
compound 17. Compound 17 is converted into a complexing ligand by
reaction with an amine, such as methyl amine to form compound
18.
Yet another exemplary synthetic scheme is set forth in Scheme 4
(FIG. 10). Compound 7 (Scheme 2) is treated with a mono-protected
diamine 21, to form compound 23. An excess of the diamine is
utilized to ensure that each of the available reactive groups is
converted to the corresponding amide. It will be apparent to those
of skill in the art that the degree of reactive group conversion
can be controlled by altering conditions, such as the concentration
of the reactants and the stoichiometry of compound 21 to compound
2.
A linker arm can also be attached to the backbone of the chelating
agent. For example, in Scheme 5 (FIG. 11), a chelator having an
amine terminated linker arm integral to the backbone is
prepared.
The above-recited synthetic schemes are intended to be exemplary of
certain embodiments of the invention, those of skill in the art
will recognize that many other synthetic strategies for producing
the ligands of the invention are available without resort to undue
experimentation.
The substituents on the isophthamidyl group and the on the backbone
joining the isophthamidyl groups can themselves comprise chelating
agents other than a hydroxyisophthamidyl group. Preferably, these
chelators comprise a plurality of anionic groups such as
carboxylate or phosphonate groups. In a preferred embodiment, these
non-PL chelating agents are selected from compounds which
themselves are capable of functioning as lanthanide chelators. In
another preferred embodiment, the chelators are aminocarboylates
(i.e. EDTA, DTPA, DOTA, NTA, HDTA, etc. and their phosphonate
analogs such as DTPP, EDTP, HDTP, NTP, etc).
Many useful chelating groups, crown ethers, cryptands and the like
are known in the art and can be incorporated into the compounds of
the invention. See, for example, Pitt et al., "The Design of
Chelating Agents for the Treatment of Iron Overload," In, INORGANIC
CHEMISTRY IN BIOLOGY AND MEDICINE; Martell, Ed.; American Chemical
Society, Washington, D.C., 1980, pp. 279-312; Lindoy, THE CHEMISTRY
OF MACROCYCLIC LIGAND COMPLEXES; Cambridge University Press,
Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY; Springer-Verlag, New
York, 1989, and references contained therein.
Additionally, a manifold of routes allowing the attachment of
chelating agents, crown ethers and cyclodextrins to other molecules
is available to those of skill in the art. See, for example, Meares
et al., "Properties of In Vivo Chelate-Tagged Proteins and
Polypeptides." In, MODIFICATIONS OF PROTEINS: FOOD, NUTRITIONAL,
AND PHARMACOLOGICAL ASPECTS;" Feeney, et al., Eds., American
Chemical Society, Washington, D.C., 1982, pp. 370-387; Kasina et
al., Bioconjugate Chem., 9: 108-117 (1998); Song et al.,
Bioconjugate Chem., 8: 249-255 (1997).
In other embodiments substituents on the isophthalamidyl group or
on the backbone are fluorescence sensitizers. Exemplary sensitizers
include rhodamine 560, 575 and 590 fluoresceins, 2- or
4-quinolones, 2 or 4-coumarins, or derivatives thereof e.g.
coumarin 445, 450, 490, 500 and 503, 4-trifluoromethylcoumarin
(TFC), 7-diethyl-amino-cumarin-3-carbohyddzide, etc., and
especially carbostyril 124 (7-amino-4-methyl-2-quinolone), coumarin
120 (7-amino-4-methyl-2-coumarin), coumarin 124
(7-amino-4-(trifluoromethyl)-2-coumarin),
aminomethyltrimethylpsoralen, napthalene and the like.
In a preferred embodiment, the sensitizer is a moiety that
comprises a napthyl moiety.
After the PL is formed and purified, the fluorescent lanthanide
complex is synthesized by any of a wide range of art-recognized
methods, including, for example, by incubating a salt of the
chelate with a lanthanide salt such as the lanthanide trihalide,
triacetate, and the like.
The compounds of the invention, in their unconjugated form are
useful as probes, indicators, separation media, and the like.
Moreover, the compounds of the invention can be conjugated to a
wide variety of compounds to create specific labels, probes,
diagnostic and/or therapeutic reagents, etc. Examples of species to
which the compounds of the invention can be conjugated include, for
example, biomolecules such as proteins (e.g., antibodies, enzymes,
receptors, etc.), nucleic acids (e g., RNA, DNA, etc.), bioactive
molecules (e.g., drugs, toxins, etc.); solid subtrates such as
glass or polymeric beads, sheets, fibers, membranes (e.g. nylon,
nitrocellulose), slides (e.g. glass, quartz) and probes; etc.
In a preferred embodiment, the species to which the compound is
conjugated is a biomolecule. Preferred biomolecules are those
selected from the group consisting of antibodies, nucleic acids,
enzymes, haptens, carbohydrates and antigens.
Assays and PL-Bearing Probes
In another preferred embodiment, the present invention provides a
PL that is tethered to another molecule, such as a probe molecule
and assays using these probes.
Assays
The following discussion is generally relevant to the assays
described herein. This discussion is intended to illustrate the
invention by reference to certain preferred embodiments and should
not be interpreted as limiting the scope of probes and assay types
in which the compounds of the invention find use. Other assay
formats utilizing the compounds of the invention will be apparent
to those of skill in the art.
Assays based on specific binding reactions are used for detecting a
wide variety of substances such as drugs, hormones, enzymes,
proteins, antibodies, and infectious agents in various biological
fluids and tissue samples. In general, the assays consist of an
analyte, a recognition moiety for the analyte, and a detectable
label. Competitive assay modalities generally utilize a binding
partner in addition to these components. In an exemplary
embodiment, the binding partner is a molecule that interacts with a
recognition moiety to form a complex that is inherently less stable
than a similar complex formed between the recognition moiety and
the analyte, and is subsequently displaced by the incoming
analyte.
Because the results of specific binding interactions are frequently
not directly observable, a variety of fluorescent labels have been
devised for determining the presence of an interaction. The
fluorophores of the invention are detected by use of fluorescence
spectroscopy or by the naked eye. An introduction to labels,
labeling procedures and detection of labels, such as are useful in
practicing the present invention, is found in Polak et al.,
INTRODUCTION TO IMMUNOCYCTOCHEMISTRY, 2.sup.nd Ed., Springer
Verlag, NY, (1977), and in Haugland, HANDBOOK OF FLUORESCENT PROBES
AND Research CHEMICALS, a combined handbook and catalogue Published
by Molecular Probes, Inc., Eugene, Oreg.(1996)
In certain embodiments, the assay is a competitive assay. In
practice, the components of the assay (i.e., recognition moiety,
binding partner and analyte) can have substantially any chemical
structure, however in a preferred embodiment, the recognition
moiety, the binding partner and the analyte are members
independently selected from the group consisting of small molecular
bioactive agents, biomolecules and combinations thereof. When a
component of the assay is a biomolecule, the biomolecule is
preferably a member selected from the group consisting of haptens,
antibodies, antigens, carbohydrates, nucleic acids, peptides,
enzymes and receptors.
In a competitive assay format, one or more than one of the
components is labeled with a compound of the invention. For
example, in one embodiment, the binding partner is labeled with a
compound of the invention and its displacement from an immobilized
recognition moiety is detected by the appearance of fluorescence in
a liquid phase of the assay. In another competitive assay format,
an immobilized enzyme is complexed with a substrate conjugated to a
compound of the invention. The complex is then contacted with a
putative antagonist. The displacement of fluorescence from the
immobilized enzyme into a liquid phase of the assay is indicative
of displacement of the substrate by the putative antagonist. These
embodiments are offered by way of example only and it will be plain
to one of skill in the art that many other competitive assay
formats can utilize and benefit from the compounds of the
invention.
In addition to ascertaining a binding event, it is frequently
desired to quantitate the magnitude of the affinity between two or
more binding partners. Thus, it is also within the scope of the
present invention to utilize the compounds disclosed herein as a
support for such assays.
Most typically, the amount of analyte present is measured by
quantitating the amount of label fixed to a binding partner,
analyte or recognition moiety following a binding event. Means of
detecting and quantitating fluorescent labels are well known to
those of skill in the art.
In another preferred embodiment, the affinity between two or more
assay constituents is measured by quantifying a population selected
from the group consisting of the analyte-recognition moiety
complex, free analyte, free binding partner, binding
partner-recognition moiety complex and combinations thereof.
The format of an assay for extracting affinity data for two
molecules can be understood by reference to an embodiment in which
a ligand that is known to bind to a receptor is displaced by an
antagonist to that receptor. Other variations on this format will
be apparent to those of skill in the art. The competitive format is
well known to those of skill in the art. See, for example, U.S.
Pat. Nos. 3,654,090 and 3,850,752.
The binding of an antagonist to a receptor can be assayed by a
competitive binding method using a ligand for that receptor and the
antagonist. The binding assay can be performed, for example, in a
96-well filtration plate assembly (Millipore Corporation, Bedford,
Mass.). One of the three binding partners (ie., the ligand,
antagonist or receptor) is generally bound to the well or to a
particulate material contained within the well.
Competition binding data can be analyzed by a number of techniques,
including nonlinear least-squares curve fitting procedure. When the
ligand is an antagonist for the receptor, this method provides the
IC50 of the antagonist (concentration of the antagonist which
inhibits specific binding of the ligand by 50% at equilibrium). The
IC50 is related to the equilibrium dissociation constant (Ki) of
the antagonist based on the Cheng and Prusoff equation:
Ki=IC50(1+L/Kd), where L is the concentration of the ligand used in
the competitive binding assay, and Kd is the dissociation constant
of the ligand as determined by Scatchard analysis. These assays are
described, among other places, in Maddox el al., J Exp Med., 158:
1211 (1983); Hampton et al., SEROLOGICAL METHODS , A LABORATORY
MANUAL, APS Press, St. Paul, Minn., 1990.
The assays of the invention can be practiced with some or all
components in solution. Alternatively, one or more components can
be substantially insoluble in the assay medium. In a preferred
embodiment, one or more members selected from the group consisting
of the recognition moiety, the binding partner and the analyte are
attached to a surface. Useful surface include, but are not limited
to, glass or polymeric beads, sheets, fibers, membranes (e.g.
nylon, nitrocellulose), slides (e.g. glass, quartz) and the
like.
The assay can be performed in a large variety of ways. It is within
the abilities of one of skill in the art to choose, for example,
when to form the fluorescent complex by chelating the lanthanide,
which assay component the chelate should be attached to and the
like. In a preferred embodiment, the fluorescent complex is formed
prior to displacing the binding partner from the binding
partner-recognition moiety complex. In another preferred
embodiment, the fluorescent complex is formed after displacing the
binding partner from the binding partner-recognition moiety
complex.
Following the displacement of the binding partner from the binding
partner-recognition moiety complex, the remaining steps of the
assay can be performed on the mixture that is formed by the
displacement or one or more of the components of the mixture can be
removed. In a preferred embodiment, the method further comprises
separating the free binding partner from a member of the group
consisting of the recognition-binding partner pair, the
analyte-recognition moiety pair and combinations thereof.
In a preferred embodiment, the assays of the invention are
immunological assays. Immlunological assays involve reactions
between immunoglobulins (antibodies) which are capable of binding
with specific antigenic determinants of various compounds and
materials (antigens). Other types of reactions include binding
between avidin and biotin, protein A and immunoglobulins, lectins
and sugar moieties and the like. See, for example, U.S. Pat. No.
4,313,734, issued to Leuvering; U.S. Pat. No. 4,435,504, issued to
Zuk; U.S. Pat. Nos. 4,452,901 and 4,960,691, issued to Gordon; and
U.S. Pat. No. 3,893,808, issued to Campbell.
These assay techniques provide the ability to detect both the
presence and amount of small quantities of analytes and are useful
in, for example medical diagnostics and forensic applications. In
the methods of the present invention, the analyte or its binding to
the recognition moiety is generally detected by the use of a
fluorescent label according to the invention.
Immunological assays are of three general types. In an exemplary
competitive binding assays, labeled reagents and unlabeled analyte
compounds compete for binding sites on a binding material. After an
incubation period, unbound materials are washed off and the amount
of labeled reagent bound to the site is compared to reference
amounts for determination of the analyte concentration in the
sample solution.
A second type of immunological assay is known as a sandwich assay
and generally involves contacting an analyte sample solution to a
surface comprising a first binding material immunologically
specific for that analyte. A second solution comprising a binding
material bearing a compound of the invention of the same type
(antigen or antibody) as the first binding material is then added
to the assay. The labeled binding material will bind to any analyte
which is bound to the first binding material. The assay system is
then subjected to a wash step to remove labeled binding material
which failed to bind with the analyte and the amount of labeled
material remaining is ordinarily proportional to the amount of
bound analyte.
A third type of immunological assay technique involves
agglutination reaction techniques and is exemplified by well-known
assays for blood antigens and serum types. Immunological
cross-reactivity between antibodies within serum and antigens
presented on red blood cell surfaces is indicated by the formation
of a three dimensional cross-linked network of red blood cells and
antibodies. The agglutination of the serum/red blood cell mixture
results in the formation of a pellet which can be visible to the
naked eye, via the fluorescence of a compound of the invention
attached to one or more components of the assay.
These assay procedures, enumerated above, were originally performed
according to liquid phase immunochemistry techniques wherein
enzymes and radiolabeled reactions were carried out in liquid
solution in apparatus such as microtiter plates. More recently,
techniques and procedures have been adapted for carrying out
"solid" phase assays wherein enymatic and immunological reactions
are carried out in solution on immobilizing substrates.
These types of assays, generally designated immunochromatographic
immunoassays, can be developed in any number of formats employing
principals of competitive, sandwich, or agglutination types of
assays. They can also involve either flow across or flow along the
immobilizing substrate. In general, the sandwich assays have the
greatest utility for detection of large protein analytes or
antibodies. The flow across type of assays have been used most
extensively in sandwich type assays.
An exemplary immunochromatographic sandwich immunoassay procedure
using the fluorescent agents of the invention employs a porous
surface and an agent of the invention as a visual label attached to
one member of a binding pair (e.g., antigen or antibody). The
porous surface is generally a flat sheet and is usually comprised
of either nylon, nitrocellulose, glass fiber, or the like. In a
typical immnunochromatographic format a region or small area of the
porous surface becomes a solid phase capturing surface by
immobilizing a member of a binding pair directly onto the surface
of a porous membrane or by indirectly attaching the member onto
capture particles (i.e., latex, glass,) which are immobilized on
the surface of a porous membrane. Direct immobilization of the
binding pair to a porous membrane or capture particles occur
through electrostatic interaction, (i.e., differences in ionic
charge), hydrophobic interaction, or covalent binding. Where
capture particles are used, the immobilization of capture particles
to porous membranes can also occur through the same phenomena or
through size exclusion preventing migration of the particles
through the pores or fibers of the membrane. Many other types of
assays can be run utilizing the compounds of the invention.
In a typical noncompetitive immunochromatographic assay, a test
sample of a biological fluid such as blood, serum, plasma, saliva,
urine, etc. must be in a sufficient volume and have a sufficient
concentration of analyte to allow for sufficient interaction to
occur between the analyte of interest, the labeled particles and
the capturing solid phase. In order to increase the reaction
kinetics, the concentration of particle labeled member of a binding
pair and the concentration of binding pair at the surface of the
porous membrane or capturing particles is optimized to produce as
much specific binding as possible and at the same time minimize any
nonspecific binding. The concentration of the particle labeled
member must be of a concentration that does not produce prozone
phenomena throughout the range of analyte concentrations that are
of interest. Such concentration optimization is well within the
abilities of one of skill.
Immunochromatographic assays can be in the form of strips or layers
of the multilayered materials of the invention employing a
hydrophobic support (e.g., Mylar, polystyrene. polypropylene,
glass, etc.) wherein one or more compounds of the invention or
moieties functionalized with a compound of the invention is either
fixed directly or indirectly with a binder such as glue to the
support. If it is desired, hydrophobic supports and housings can be
employed to reduce evaporation of the fluid phase while the
immunoreactants are being brought into contact with each other.
In an exemplary non-competitive assay in accordance with this
aspect of the invention, an analyte is solubilized, deposited and
bound onto the particulate material. The particulate material is
then hydrated and sequentially exposed to primary antibodies and
enzyme-conjugated secondary antibodies specific for the primary
antibodies, with washing steps in between where appropriate. Enzyme
levels are then determined by, for instance, substrate conversion
protocols well known in the art, and the amount of primary
antibodies can thus be measured by reference to a standard run in
parallel.
Additionally, a binding domain of a receptor, for example, can
serve as the focal point for a drug discovery assay, where, for
example, the receptor is immobilized, and incubated both with
agents (i.e., ligands) known to interact with the binding domain
thereof, and a quantity of a particular drug or inhibitory agent
under test. One of the incubation components is labeled with a
compound of the invention. The extent to which the drug binds with
the receptor and thereby inhibits receptor-ligand complex formation
can then be measured. Such possibilities for drug discovery assays
are contemplated herein and are considered within the scope of the
present invention. Other focal points and appropriate assay formats
will be apparent to those of skill in the art.
The compounds and methods of the invention can also be used to
sequence nucleic acids and peptides. Fluorescent-labeled
oligonucleotide primers have been used in place of radiolabeled
primers for sensitive detection of DNA fragments (U.S. Pat. No.
4,855,225 to Smith et al.). Additionally, DNA sequencing products
can be labeled with fluorescent dideoxynucleotides (U.S. Pat. No.
5,047,519 to Prober et al.) or by the direct incorporation of a
fluorescent labeled deoxynucleotide (Voss et al. Nucl. Acids Res.
17:2517 (1989)). The compounds of the invention are useful in both
of these formats. As currently practiced, fluorescent sequencing
reactions circumvent many of the problems associated with the use
of radionuclides.
As discussed above, the fluorescent complex can be formed at
substantially any step of the assay. This is equally true in those
embodiments, wherein one or more components of the assay mixture
are removed following the displacement of the binding partner. In a
preferred embodiment, the fluorescent complex is formed following
the separation.
Compounds of the invention can be used to indicate the presence and
amount of an enzyme in a mixture. For example, in certain
embodiments, Q.sup.1 is an enzymatically labile group and the
presence of the labile group on the phenolic oxygen of the
hydroxyisophthamidyl group will prevent the formation of a stable
complex of a lanthanide ion. This situation is reversed, and a
stable lanthanide complex is formed, when the hydroxyisophthamidyl
chelate is contacted with an enzyme that is capable of cleaving the
labile group, thus, freeing the phenolic oxygen anion. Similar to
the embodiments discussed above, the assay mixture can be contacted
with the enzyme at any time during the assay process. Additionally,
if a component is separated from the reaction mixture (e.g., the
liberated binding partner), the separated component and/or the
remaining component can be contacted with the enzyme.
In a preferred embodiment, wherein Q.sup.1 is an enzymatically
labile group, the method further includes contacting a member
selected from the group consisting of the binding
partner-recognition moiety complex, the free binding partner and
combinations thereof with an enzyme, thereby removing the
enzymatically labile group.
An array of enzymatically removable groups is known in the art and
it is within the abilities of one of skill in the art to select an
appropriate enzymatically labile group for a particular
application. In a preferred embodiment, the enzymatically labile
group comprises a component of a member selected from the group
consisting of phosphate, sulfate, acyl and glycoside groups.
Enzymes capable of removing these groups include, for example,
esterases, phosphatases, glycosidases and the like.
In another preferred embodiment, the removal of the enzymatically
labile group and the subsequent formation of a fluorescent complex
is used to detect the presence of an enzyme capable of removing the
enzymatically labile group. See, for example, Drevin et al., U.S.
Pat. No. 5,252,462, issued Oct. 12, 1993.
Although the compounds of the invention can be tethered to any
component of the assay, they will most generally be attached to the
binding partner. In this embodiment, the compounds of the invention
can be attached to the binding partner through a reactive group on
a phthalamidyl moiety, backbone or amide substitutent.
Alternatively, they can be attached to the binding partner through
a reactive group on the aromatic nucleus of one or more of the
hydroxyisophthamidyl, moieties of the compounds. As discussed
above, many suitable reactive groups are known to those of skill in
the art and one of skill will be able to both choose and prepare a
hydroxyisophthamidyl-chelate that is appropriately functionalized
for a particular application.
It will generally be preferred that the linkage between the
hydroxyisophthamidyl-chelates and the binding partner be stable
under the conditions of the assay. Many stable linkages can be
formed between the binding partner and the hydroxyisophthamidyl
chelate including, for example, amides, amines, ethers, ureas, and
the like. In a preferred embodiment, the linkage between the
binding partner and a compound of the invention is a member
selected from the group consisting of amide, thioamide, thoiurea
and carbamate linkages. Suitable reactive groups and linkages are
discussed in greater detail above.
In general, to determine the concentration of a target molecule,
such as, for example, a nucleic acid, it is preferable to first
obtain reference data in which constant amounts of probe and
nucleic acid ligand are contacted with varying amounts of target.
The fluorescence emission of each of the reference mixtures is used
to derive a graph or table in which target concentration is
compared to fluorescence emission. For example, a probe that: a)
hybridizes to a target-free nucleic acid ligand; and b) has a
stem-loop architecture with the 5' and 3' termini being the sites
of fluorescent group and PL labeling, could be used to obtain such
reference data. Such a probe gives a characteristic emission
profile in which the fluorescence emission decreases as the target
concentration increases in the presence of a constant amount of
probe and nucleic acid ligand. Then, a test mixture with an unknown
amount of target is contacted with the same amount of first nucleic
acid ligand and second probe, and the fluorescence emission is
determined. The value of the fluorescence emission is then compared
with the reference data to obtain the concentration of the target
in the test mixture.
Muliplex Analyses
In another preferred embodiment, the quenchers of the invention are
utilized as a component of one or more probes used in an assay
designed to detect multiple species in a mixture. An assays used to
detect two or more species by using at least two probes bearing
different fluorophores is referred to herein as a "multiplex
analysis." A schematic diagram of such a multiplex analysis using a
PL is set forth in FIG. 20.
Probes that include the compounds of the invention are also useful
in performing multiplex-type analyses and assays. In a typical
multiplex analysis, two or more distinct species (or regions of one
or more species) are detected using two or more probes, wherein
each of the probes is labeled with a different fluorophore.
Preferred multiplex analyses relying on fluorescent energy transfer
ideally meet several criteria. The fluorescent species should be
bright and spectrally well-resolved and the energy transfer between
the fluorescent species and the acceptor should be efficient.
Because of the ready availability of PLs of the invention having
different emission characteristics, the compounds of the invention
are particularly well suited for use in multiplex applications.
Access to PLs having a range of absorbance characteristics allows
for the design of FET probes in which the acceptor absorbance
properties and the PL emission properties are matched, thereby
providing a useful level of spectral overlap.
The simultaneous use of two or more probes using FET is known in
the art. For example, multiplex assays using nucleic acid probes
with different sequence specificities have been described.
Fluorescent probes have been used to determine whether an
individual is homozygous wild-type, homozygous mutant or
heterozygous for a particular mutation. For example, using one
quenched-fluorescein molecular beacon that recognizes the wild-type
sequence and another rhodamine-quenched molecular beacon that
recognizes a mutant allele, it is possible to genotype individuals
for the .beta.-chemokine receptor (Kostrikis et al. Science
279:1228-1229 (1998)). The presence of only a fluorescein signal
indicates that the individual is wild-type, and the presence of
rhodamine signal only indicates that the individual is a homozygous
mutant. The presence of both rhodamine and fluorescein signal is
diagnostic of a heterozygote. Tyagi et al. Nature Biotechnology 16:
49-53 (1998)) have described the simultaneous use of four
differently labeled molecular beacons for allele discrimination,
and Lee et al., BioTechniques 27: 342-349 (1999) have described
seven color homogenous detection of six PCR products.
The PLs of the present invention can be used in multiplex assays
designed to detect and/or quantify substantially any species,
including, for example, whole cells, viruses, proteins (e.g.,
enzymes, antibodies, receptors), glycoproteins, lipoproteins,
subcellular particles, organisms (e.g., Salmonella), nucleic acids
(e.g., DNA, RNA, and analogues thereof), polysaccharides,
lipopolysaccharides, lipids, fatty acids, non-biological polymers
and small bioactive molecules (e.g., toxins, drugs, pesticides,
metabolites, hormones, alkaloids, steroids).
Recognition Moieties
As used herein, the term "recognition moiety" refers to molecules
that can interact with an analyte via either attractive or
repulsive mechanisms. In a preferred embodiment, a recognition
moiety is conjugated to a compound of the invention. In another
exemplary embodiment, the analyte and the recognition moiety form
an intimately associated pair by, for example, covalent bonding,
ionic bonding, ion pairing, van der Waals association and the like.
In another exemplary embodiment, the analyte and recognition moiety
interact by a repulsive mechanism such as incompatible steric
characteristics, charge-charge repulsion, hydrophilic-hydrophobic
interactions and the like. It is understood that there is overlap
between the generic terms "recognition moiety" and "analyte." In a
particular application, a species may be an analyte, while in a
different application, the species serves as a recognition moiety.
In certain embodiments, the compounds of the invention serve as
recognition moieties (e.g., when the analyte is a metal ion).
Recognition moieties can be selected from a wide range of small
bioactive molecules (e.g., drugs, pesticides, toxins, etc.),
organic functional groups (e.g., amines, carbonyls, carboxylates,
etc.), biomolecules, metals, metal chelates and organometallic
compounds.
When the recognition moiety is an amine, in preferred embodiments,
the recognition moiety will interact with a structure on the
analyte which reacts by interacting (e.g., binding, complexing)
with the amine (e.g. carbonyl groups, alkylhalo groups). In another
preferred embodiment, the amine is protonated by an acidic moiety
on the analyte of interest (e.g., carboxylic acid, sulfonic
acid).
In certain preferred embodiments, when the recognition moiety is a
carboxylic acid, the recognition moiety will interact with the
analyte by, for example, complexation (e.g., metal ions). In still
other preferred embodiments, the carboxylic acid will protonate a
basic group on the analyte (e.g. amine).
In another preferred embodiment, the recognition moiety is a drug
moiety. The drug moieties can be agents already accepted for
clinical use or they can be drugs whose use is experimental, or
whose activity or mechanism of action is under investigation. The
drug moieties can have a proven action in a given disease state or
can be only hypothesized to show desirable action in a given
disease state. In a preferred embodiment, the drug moieties are
compounds which are being screened for their ability to interact
with an analyte of choice. As such, drug moieties which are useful
as recognition moieties in the instant invention include drugs from
a broad range of drug classes having a variety of pharmacological
activities.
Classes of useful agents include, for example, non-steroidal
anti-inflammatory drugs (NSAIDS). The NSAIDS can, for example, be
selected from the following categories: (e.g., propionic acid
derivatives, acetic acid derivatives, fenamic acid derivatives,
biphenylcarboxylic acid derivatives and oxicams); steroidal
anti-inflammatory drugs including hydrocortisone and the like;
antihistaminic drugs (e.g., chlorpheniramine, triprolidine);
antitussive drugs (e.g., dextromethorphan, codeine, carmiphen and
carbetapentane); antipruritic drugs (e.g., methidilizine and
trimeprizine); anticholinergic drugs (e.g., scopolamine, atropine,
homatropine, levodopa); anti-emetic and antinauseant drugs (e.g.,
cyclizine, meclizine, chlorpromazine, buclizine); anorexic drugs
(e.g., benzphetamine, phentermine, chlorphentermine, fenfluramine);
central stimulant drugs (e.g., amphetamine, methamphetamine,
dextroamphetamine and methylphenidate); antiarrhythmic drugs (e.g.,
propanolol, procainamide, disopyraminde, quinidine, encainide);
.beta.-adrenergic blocker drugs (e.g., metoprolol, acebutolol,
betaxolol, labetalol and timolol); cardiotonic drugs (e.g.,
milrinone, amrinone and dobutamine); antihypertensive drugs (e.g.,
enalapril, clonidine, hydralazine, minoxidil, guanadrel,
guanethidine);diuretic drugs (e.g., amiloride and
hydrochlorothiazide); vasodilator drugs (e.g., diltazem,
amiodarone, isosuprine, nylidrin, tolazoline and verapamil);
vasoconstrictor drugs (e.g., dihydroergotamine, ergotamine and
methylsergide); antiulcer drugs (e.g., ranitidine and cimetidine);
anesthetic drugs (e.g., lidocaine, bupivacaine, chlorprocaine,
dibucaine); antidepressant drugs (e.g., imipramine, desipramine,
amitryptiline, nortryptiline); tranquilizer and sedative drugs
(e.g., chlordiazepoxide, benacytyzine, benzquinamide, flurazapam,
hydroxyzine, loxapine and promazine); antipsychotic drugs (e.g.,
chlorprothixene, fluphenazine, haloperidol, molindone, thioridazine
and trifluoperazine); antimicrobial drugs (antibacterial,
antifungal, antiprotozoal and antiviral drugs).
Antimicrobial drugs which are preferred for incorporation into the
present composition include, for example, pharmaceutically
acceptable salts of .beta.-lactam drugs, quinolone drugs,
ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin,
triclosan, doxycycline, capreomycin, chlorhexidine,
chlortetracycline, oxytetracycline, clindamycin, ethambutol,
hexamidine isothionate, metronidazole, pentamidine, gentamycin,
kanamycin, lineomycin, methacycline, methenamine, minocycline,
neomycin, netilmycin, paromomycin, streptomycin, tobramycin,
miconazole and amanfadine.
Other drug moieties of use in practicing the present invention
include antineoplastic drugs (e.g., antiandrogens (e.g., leuprolide
or flutamide), cytocidal agents (e.g., adriamycin, doxorubicin,
taxol, cyclophosphamide, busulfan, cisplatin, .alpha.-2-interferon)
anti-estrogens (e.g., tamoxifen), antimetabolites (e.g.,
fluorouracil, methotrexate, mercaptopurine, thioguanine).
The recognition moiety can also comprise hormones (e.g.,
medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide
or somatostatin); muscle relaxant drugs (e.g., cinnamedrine,
cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine,
idaverine, ritodrine, dephenoxylate, dantrolene and azumolen);
antispasmodic drugs; bone-active drugs (e.g., diphosphonate and
phosphonoalkylphosphinate drug compounds); endocrine modulating
drugs (e.g., contraceptives (e.g., ethinodiol, ethinyl estradiol,
norethindrone, mestranol, desogestrel, medroxyprogesterone),
modulators of diabetes (e.g., glyburide or chlorpropamide),
anabolics, such as testolactone or stanozolol, androgens (e.g.,
methyltestosterone, testosterone or fluoxymesterone), antidiuretics
(e.g., desmopressin) and calcitonins).
Also of use in the present invention are estrogens (e.g.,
diethylstilbesterol), glucocorticoids (e.g., triamcinolone,
betamethasone, etc.) and progenstogens, such as norethindrone,
ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,
liothyronine or levothyroxine) or anti-thyroid agents (e.g.
methimazole); antihyperprolactinemic drugs (e.g., cabergoline);
hormone suppressors (e.g., danazol or goserelin), oxytocics (e.g.,
methylergonovine or oxytocin) and prostaglandins, such as
mioprostol, alprostadil or dinoprostone, can also be employed.
Other useful recognition moieties include immunomodulating drugs
(e.g., antihistamines, mast cell stabilizers, such as lodoxamide
and/or cromolyn, steroids (e.g., triamcinolone, beclomethazone,
cortisone, dexamethasone, prednisolone, methylprednisolone,
beclomethasone, or clobetasol), histamine H.sub.2 antagonists
(e.g., famotidine, cimetidine, ranitidine), immunosuppressants
(e.g., azathioprine, cyclosporin), etc. Groups with
anti-inflammatory activity, such as sulindac, etodolac, ketoprofen
and ketorolac, are also of use. Other drugs of use in conjunction
with the present invention will be apparent to those of skill in
the art.
The above enumerated, and other molecules, can be attached to the
compounds of the invention, to solid substrates and the like by
methods well-known to those of skill in the art. Ample guidance can
be found in literature devoted to, for example, the fields of
bioconjugate chemistry and drug delivery. For example, one of
skill, faced with a drug comprising an available amine will be able
to choose from among a variety of amine derivatizing reactions,
locate an appropriately functionalized partner (e.g., a carboxylic
acid terminated thiol) for the organic layer and react the partners
under conditions chosen to effect the desired coupling (e.g.,
dehydrating agents, e.g., dicyclohexylcarbodiimide). See, for
example, MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND
PHARMACOLOGICAL ASPECTS, Feeney et al., Eds., American Chemical
Society, Washington, D.C., 1982, pp. 370-387; POLYMERIC DRUGS AND
DRUG DELIVERY SYSTEMS, Dunn et al., Eds., American Chemical
Society, Washington, D.C., 1991.
When the recognition moiety is a chelating agent, crown ether or
cyclodextrin, host-guest chemistry will dominate the interaction
between the recognition moiety and the analyte. The use of
host-guest chemistry allows a great degree of recognition
moiety-analyte specificity to be engineered into a compound or
assay of the invention. The use of these compounds to bind to
specific compounds is well known to those of skill in the art. See,
for example, Pitt et al., "The Design of Chelating Agents for the
Treatment of Iron Overload," In, INORGANIC CHEMISTRY IN BIOLOGY AND
MEDICINE; Martell, Ed.; American Chemical Society, Washington,
D.C., 1980, pp. 279-312; Lindoy, THE CHEMISTRY OF MACROCYCLIC
LIGAND COMPLEXES; Cambridge University Press, Cambridge, 1989;
Dugas, BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, and
references contained therein.
Additionally, a manifold of routes allowing the attachment of
chelating agents, crown ethers and cyclodextrins to other molecules
is available to those of skill in the art. See, for example, Meares
et al., "Properties of In Vivo Chelate-Tagged Proteins and
Polypeptides." In, MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND
PHARAMACOLOGICAL ASPECTS;" Feeney, et al., Eds., American Chemical
Society, Washington, D.C., 1982, pp. 370-387; Kasina et al.,
Bioconjugate Chem., 9: 108-117 (1998); Song et al., Bioconjugate
Chem., 8: 249-255 (1997).
In another preferred embodiment, the recognition moiety forms an
inclusion complex with the analyte of interest. In a particularly
preferred embodiment, the recognition moiety is a cyclodextrin or
modified cyclodextrin. Cyclodextrins are a group of cyclic
oligosaccharides produced by numerous microorganisms. Cyclodextrins
have a ring structure which has a basket-like shape. This shape
allows cyclodextrins to include many kinds of molecules into their
internal cavity. See, for example, Szejtli, CYCLODEXTRINS AND THEIR
INCLUSION COMPLEXES; Akademiai Klado, Budapest, 1982; and Bender et
al., CYCLODEXTRIN CHEMISTRY, Springer-Verlag, Berlin, 1978.
Cyclodextrins are able to form inclusion complexes with an array of
bioactive molecules including, for example, drugs, pesticides,
herbicides and agents of war. See, Tenjarla et al., J. Pharm. Sci.,
87: 425-429 (1998); Zughul et al., Pharm. Dev. Technol., 3: 43-53
(1998); and Albers et al., Crit. Rev. Ther. Drug Carrier Syst., 12:
311-337 (1995). Importantly, cyclodextrins are able to discriminate
between enantiomers of compounds in their inclusion complexes.
Thus, in one preferred embodiment, the invention provides for the
detection of a particular enantiomer in a mixture of enantiomers.
See, Koppenhoefer et al., J. Chromatogr., A 793: 153-164
(1998).
The cyclodextrin or any other recognition moiety can be attached to
a compound of the invention, solid support and the like either
directly or through a spacer arm. See, Yamamoto et al., J. Phys.
Chem. B, 101: 6855-6860 (1997). Methods to attach cyclodextrins to
other molecules are well known to those of skill in the
chromatographic and pharmaceutical arts. See, Sreenivasan, K. J.,
Appl. Polym. Sci., 60: 2245-2249 (1996).
In another exemplary embodiment, the recognition moiety is a
polyaminocarboxylate chelating agent, such as
ethylenediaminetetraacetic acid (EDTA) or
diethylenetriaminepentaacetic acid (DTPA). These recognition
moieties can be attached, for example, to any amine-terminated
component of a compound of the invention, solid support or a spacer
arm, for example, by utilizing the commercially available
dianhydride (Aldrich Chemical Co., Milwaukee, Wis.).
In still further preferred embodiments, the recognition moiety is a
biomolecule such as a protein, nucleic acid, peptide or an
antibody. Biomolecules useful in practicing the present invention
can be derived from any source. The biomolecules can be isolated
from natural sources or can be produced by synthetic methods.
Proteins can be natural proteins or mutated proteins. Mutations can
be effected by chemical mutagenesis, site-directed mutagenesis or
other means of inducing mutations known to those of skill in the
art. Proteins useful in practicing the instant invention include,
for example, enzymes, antigens, antibodies and receptors.
Antibodies can be either polyclonal or monoclonal. Peptides and
nucleic acids can be isolated from natural sources or can be wholly
or partially synthetic in origin.
In those embodiments wherein the recognition moiety is a protein or
antibody, the protein can be tethered to a compound of the
invention, solid support or a crosslinking agent by any reactive
peptide residue available on the surface of the protein. In
preferred embodiments, the reactive groups are amines or
carboxylates. In particularly preferred embodiments, the reactive
groups are the .epsilon.-amine groups of lysine residues.
Recognition moieties which are antibodies can be used to recognize
analytes which are proteins, peptides, nucleic acids, saccharides
or small bioactive materials, such as drugs, herbicides,
pesticides, industrial chemicals and agents of war. Methods of
raising antibodies for specific molecules are well-known to those
of skill in the art. See, U.S. Pat. No. 5,147,786, issued to Feng
et al. on Sep. 15, 1992; U.S. Pat. No. 5,334,528, issued to Stanker
et al. on Aug. 2, 1994; U.S. Pat. No. 5,686,237, issued to
Al-Bayati, M. A. S. on Nov. 11, 1997; and U.S. Pat. No. 5,573,922,
issued to Hoess et al. on Nov. 12, 1996. Methods for attaching
antibodies agents to surfaces are also known in the art. See,
Delamarche et al. Langmuir, 12: 1944-1946 (1996).
A recognition moiety can be conjugated to a compound of the
invention by any of a large number of art-known attachment methods,
as discussed above. In one embodiment, the recognition moiety is
tethered directly to the hydroxyisophthamidyl chelate through a
group on the aromatic hydroxyisophthamidyl nucleus, backbone or
amide substituent. In another exemplary embodiment, a reactive
bifunctional crosslinking agent is attached reactive group on a PL
and this conjugate is subsequently bound to the recognition moiety
via the reactive group on the crosslinking component and a group of
complementary reactivity on the recognition moiety. Many useful
crosslinking agents can be purchased commercially (Pierce Rockford,
Ill.) or can be synthesized using techniques known in the art.
Alternatively, the recognition moiety and cross-linking agent are
coupled prior to attaching the hydroxyisophthamidyl chelate to the
recognition moiety.
Analytes
The materials and methods of the present invention can be used to
detect any analyte, or class of analytes, which interact with a
recognition moiety in a detectable manner. The interaction between
the analyte and recognition moiety can be any physicochemical
interaction, including covalent bonding, ionic bonding, hydrogen
bonding, van der Waals interactions, repulsive electronic
interactions, attractive electronic interactions and
hydrophobic/hydrophilic interactions.
In a preferred embodiment, the interaction is an ionic interaction.
In this embodiment, an acid, base, metal ion or metal ion-binding
ligand is the analyte. In a still further preferred embodiment, the
interaction is a hydrogen bonding interaction. In particularly
preferred embodiments, the hybridization of a nucleic acid to a
nucleic acid having a complementary sequence is detected. In
another preferred embodiment, the interaction is between an enzyme
or receptor and a small molecule or peptide which binds
thereto.
In another embodiment, the analyte competes for the recognition
moiety with another agent which has been bound to the recognition
moiety prior to introducing the analyte of interest. In this
embodiment, it is the process or result of the analyte displacing
the pre-bound agent which causes the detectable levels of
fluorescence from the compound of the invention. Suitable
combinations of recognition moieties and analytes will be apparent
to those of skill in the art.
In presently preferred embodiments, the analyte is a member
selected from the group consisting of acids, bases, organic ions,
inorganic ions, pharmaceuticals, herbicides, pesticides and
biomolecules. Each of these agents, where practicable, can be
detected as a vapor or a liquid. These agents can be present as
components in mixtures of structurally unrelated compounds, racemic
mixtures of stereoisomers, non-racemic mixtures of stereoisomers,
mixtures of diastereomers, mixtures of positional isomers or as
pure compounds. Within the scope of the invention is a device and a
method to detect a particular analyte of interest without
interference from other substances within a mixture.
Organic ions which are substantially non-acidic and non-basic
(e.g., quaternary alkylammonium salts) can be detected by a labeled
recognition moiety of the invention. For example, a PL-labeled
recognition moiety with ion exchange properties is useful in the
present invention. A specific example is the exchange of a cation
such as dodecyltrimethylammonium cation for a metal ion such as
sodium. Recognition moieties that form inclusion complexes with
organic ions are also of use. For example, crown ethers and
cryptands can be used to form inclusion complexes with organic ions
such as quaternary ammonium cations.
Inorganic ions such as metal ions and complex ions (e.g.,
SO.sub.4.sup.-2, PO.sub.4.sup.-3) can also be detected using the
PLs and methods of the invention. Metal ions can be detected, for
example, by their complexation or chelation by PLs or chelating
agents bound to a compound of the invention. In this embodiment,
the recognition moiety can be a simple monovalent moiety (e.g.,
carboxylate, amine, thiol) or can be a more structurally complex
agent (e.g., ethylenediaminepentaacetic acid, crown ethers, aza
crowns, thia crowns).
Complex inorganic ions can be detected by their ability to compete
with PLs for bound metal ions in ligand-metal complexes. When a
ligand bound to a PL forms a metal-complex having a thermodynamic
stability constant which is less than that of the complex between
the metal and the complex ion, the complex ion will cause the
dissociation of the metal ion from the immobilized ligand. If the
metal ion is the complexed lanthanide, the fluorescence will be
decreased. Methods of determining stability constants for compounds
formed between metal ions and ligands are well known to those of
skill in the art. Using these stability constants, chelates that
are specific for particular ions can be manufactured. See, Martell,
A. E., Motekaitis, R. J., DETERMINATION AND USE OF STABILITY
CONSTANTS, 2d Ed., VCH Publishers, New York 1992.
In a preferred embodiment, the affinity of an analyte for a
particular metal ion is exploited by using a compound of the
invention that includes that particular metal ion. The metal ion
generally must have available at least one empty coordination site
to which the analyte can bind. Alternatively, at least one bond
between the metal and the metal-immobilizing agent must be
sufficiently labile in the presence of the analyte to allow the
displacement of at least one bond of the immobilizing reagent by
the analyte. The interaction between the analyte and the metal ion
can be detected using a number of art-recognized techniques,
including, for example, UV/V is and fluorescence spectroscopy.
Other combinations of analytes and recognition moieties will be
apparent to those of skill in the art.
Probes
The invention provides probes including PL moieties conjugated to,
for example, a target species, a ligand for a target species (e.g.,
nucleic acid, peptide, etc.), a small molecule (e.g., drug,
pesticide, etc.), and the like.
Nucleic Acid Probes
The PLs of the invention are useful in conjunction with
nucleic-acid probes and they can be used as components of detection
agents in a variety of DNA amplification/quantification strategies
including, for example, 5'-nuclease assay, Strand Displacement
Amplification (SDA), Nucleic Acid Sequence-Based Amplification
(NASBA), Rolling Circle Amplification (RCA), as well as for direct
detection of targets in solution phase or solid phase (e.g., array)
assays. Furthermore, the PL-derivatized nucleic acids can be used
in probes of substantially any format, including, for example,
format selected from molecular beacons, scorpion probes, sunrise
probes, conformationally assisted probes, light up probes and
TaqMan.TM. probes.
Thus in a further aspect, the present invention provides a method
for detecting a nucleic acid target sequence. The method includes:
(a) contacting the target sequence with a detector nucleic acid;
(b) hybridizing the target binding sequence to the target sequence,
thereby altering the conformation of the detector nucleic acid,
causing a change in a fluorescence parameter; and (c) detecting the
change in the fluorescence parameter, thereby detecting the nucleic
acid target sequence.
In the methods described herein, unless otherwise noted, a
preferred detector nucleic acid includes a single-stranded target
binding sequence. The binding sequence has linked thereto: i) a
fluorophore; and ii) a PL of the invention. Moreover, prior to its
hybridization to a complementary sequence, the detector nucleic
acid is preferably in a conformation that allows fluorescence
energy transfer between the fluorophore and the PL when the
fluorophore is excited. Furthermore, in each of the methods
described in this section, a change in fluorescence is detected as
an indication of the presence of the target sequence, and that
change in fluorescence is preferably detected in-real time.
In another aspect, the invention provides a further method for
detecting the presence of a nucleic acid target sequence. The
method includes: (a) hybridizing to the target sequence a detector
nucleic acid comprising a single-stranded target binding sequence
and an intramolecularly associated secondary structure 5' to the
target binding sequence, wherein at least a portion of the target
sequence forms a single stranded tail which is available for
hybridization to the target sequence; (b) in a primer extension
reaction, synthesizing a complementary strand using the
intramolecularly associated secondary structure as a template,
thereby dissociating the intramolecularly associated secondary
structure and producing a change in a fluorescence parameter; (c)
detecting the change in the fluorescence parameter, thereby
detecting the nucleic acid target sequence.
In this method, and unless otherwise noted, the other methods
described in this section, the detector nucleic acid can assume
substantially any intramolecularly associated secondary structure,
but this structure is preferably a member selected from hairpins,
stem-loop structures, pseudoknots, triple helices and
conformationally assisted structures. Moreover, the
intramolecularly base-paired secondary structure preferably
comprises a portion of the target binding sequence. Moreover, the
intramolecularly associated secondary structure preferably includes
a totally or partially single-stranded endonuclease recognition
site.
The complementary strand can be prepared by any art-recognized
method for preparing such strands, but is preferably synthesized in
a target amplification reaction, and more preferably by extension
of the target sequence using the detector nucleic acid as a
template.
In another aspect, the invention provides a method for detecting
amplification of a target sequence. The method includes the use of
an amplification reaction including the following steps: (a)
hybridizing the target sequence and a detector nucleic acid. The
detector nucleic acid includes a single-stranded target binding
sequence and an intramolecularly associated secondary structure 5'
to the target binding sequence. At least a portion of the target
sequence forms a single stranded tail which is available for
hybridization to the target sequence; (b) extending the hybridized
detector nucleic acid on the target sequence with a polymerase to
produce a detector nucleic acid extension product and separating
the detector nucleic acid extension product from the target
sequence; (c) hybridizing a primer to the detector nucleic acid
extension product and extending the primer with the polymerase,
thereby linearizing the intramolecularly associated secondary
structure and producing a change in a fluorescence parameter; and
(d) detecting the change in the fluorescence parameter, thereby
detecting the target sequence.
In yet a further aspect, the invention provides a method of
ascertaining whether a first nucleic acid and a second nucleic acid
hybridize. In this method, the first nucleic acid includes a PL
according to the invention. The method includes: (a) contacting the
first nucleic acid with the second nucleic acid; (b) detecting an
alteration in a fluorescent property of a member selected from the
first nucleic acid, the second nucleic acid and a combination
thereof, thereby ascertaining whether the hybridization occurs.
A probe bearing both a PL and a fluorophore can be used or,
alternatively, one or more of the nucleic acids can be singly
labeled with a PL or fluorophore. When a nucleic acid singly
labeled with a PL is the probe, the interaction between the first
and second nucleic acids can be detected by observing the quenching
of the native nucleic acid fluorescence or, more preferably, the
quenching of the fluorescence of a fluorophore attached to the
second nucleic acid.
In addition to their general utility in species designed to probe
nucleic acid amplification, detection and quantification, the
present PLs can be used in substantially any nucleic acid probe
format now known or later discovered. For example, the PLs of the
invention can be incorporated into probe motifs, such as Taqman
probes (Held et al., Genome Res. 6: 986-994 (1996), Holland et al.,
Proc. Nat. Acad. Sci. USA 88: 7276-7280 (1991), Lee et al., Nucleic
Acids Res. 21: 3761-3766 (1993)), molecular beacons (Tyagi et al.,
Nature Biotechnology 14:303-308 (1996), Jayasena et al., U.S. Pat.
No. 5,989,823, issued Nov. 23, 1999)) scorpion probes (Whitcomb et
al., Nature Biotechnology 17: 804-807 (1999)), sunrise probes
(Nazarenko et al., Nucleic Acids Res. 25: 2516-2521 (1997)),
conformationally assisted probes (Cook, R., copending and commonly
assigned U.S. Provisional Application 60/138,376, filed Jun. 9,
1999), peptide nucleic acid (PNA)-based light up probes (Kubista et
al., WO 97/45539, December 1997), double-strand specific DNA dyes
(Higuchi et al, BioTechnology 10: 413-417 (1992), Wittwer et al,
BioTechniques 22: 130-138 (1997)) and the like. These and other
probe motifs with which the present PLs can be used are reviewed in
NONISOTOPIC DNA PROBE TECHNIQUES, Academic Press, Inc. 1992.
The nucleic acids for use in the probes of the invention can be any
suitable size, and are preferably in the range of from about 10 to
about 100 nucleotides, more preferably from about 10 to about 80
nucleotides and more preferably still, from about 20 to about 40
nucleotides. The precise sequence and length of a nucleic acid
probe of the invention depends in part on the nature of the target
polynucleotide to which it binds. The binding location and length
may be varied to achieve appropriate annealing and melting
properties for a particular embodiment. Guidance for making such
design choices can be found in many art-recognized references.
Preferably, the 3'-terminal nucleotide of the nucleic acid probe is
blocked or rendered incapable of extension by a nucleic acid
polymerase. Such blocking is conveniently carried out by the
attachment of a donor or acceptor molecule to the terminal
3'-position of the nucleic acid probe by a linking moiety.
The nucleic acid can comprise DNA, RNA or chimeric mixtures or
derivatives or modified versions thereof. Both the probe and target
nucleic acid can be present as a single strand, duplex, triplex,
etc. In addition to being labeled with an molecular energy transfer
donor and a molecular energy transfer acceptor moiety, the nucleic
acid can be modified at the base moiety, sugar moiety, or phosphate
backbone with other groups such as radioactive labels, minor groove
binders, intercalating agents an the like.
For example, the nucleic acid can comprise at least one modified
base moiety which is selected from the group including, but not
limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N.sup.6 -isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N.sup.6 -adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N.sup.6 -isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methyl ester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and
2,6-diaminopurine.
In another embodiment, the nucleic acid comprises at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the nucleic acid comprises at least one
modified phosphate backbone selected from the group including, but
not limited to, a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
Phosphodiester linked nucleic acids of the invention can be
synthesized by standard methods known in the art, e.g. by use of an
automated DNA synthesizer (such as are commercially available from
P.E. Biosystems, etc.) using commercially available amidite
chemistries. Nucleic acids bearing modified phosphodiester linking
groups can be synthesized by methods known in the art. For example,
phosphorothioate nucleic acids may be synthesized by the method of
Stein et al. (Nucl. Acids Res. 16:3209 (1988)), methylphosphonate
nucleic acids can be prepared by use of controlled pore glass
polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451 (1988)). Other methods of synthesizing both
phosphodiester- and modified phosphodiester-linked nucleic acids
will be apparent to those of skill in the art.
Nucleic acid probes of the invention can be synthesized by a number
of approaches, e.g., Ozaki et al., Nucleic Acids Research, 20:
5205-5214 (1992); Agrawal et al., Nucleic Acids Research, 18:
5419-5423 (1990); or the like. The nucleic acid probes of the
invention are conveniently synthesized on an automated DNA
synthesizer, e.g. a P.E. Biosystems, Inc. (Foster City, Calif.)
model 392 or 394 DNA/RNA Synthesizer, using standard chemistries,
such as phosphoramidite chemistry (see, for example, disclosed in
the following references: Beaucage et al., Tetrahedron, 48:
2223-2311 (1992); Molko et al., U.S. Pat. No. 4,980,460; Koster et
al., U.S. Pat. No. 4,725,677; Caruthers et al., U.S. Pat. Nos.
4,415,732; 4,458,066; and 4,973,679. Alternative chemistries
resulting in non-natural backbone groups, such as phosphorothioate,
phosphoramidate, and the like, can also be employed.
When the nucleic acids are synthesized utilizing an automated
nucleic acid synthesizer, the stabilizing moiety, energy transfer
donor and energy transfer acceptor moieties are preferably
introduced during automated synthesis. Alternatively, one or more
of these moieties can be introduced either before or after the
automated synthesis procedure has commenced. In another exemplary
embodiment, one or more of these moieties is introduced after the
automated synthesis is complete.
The donor moiety is preferably separated from the PL by at least
about 10 nucleotides, and more preferably by at least about 15
nucleotides. The donor moiety is preferably attached to either the
3'- or 5'-terminal nucleotides of the probe. The PL moiety is also
preferably attached to either the 3'- or 5'-terminal nucleotides of
the probe. More preferably, the donor and acceptor moieties are
attached to the 3'- and 5'- or 5'- and 3'-terminal nucleotides of
the probe, respectively.
Once the desired nucleic acid is synthesized, it is preferably
cleaved from the solid support on which it was synthesized and
treated, by methods known in the art, to remove any protecting
groups present (e.g., 60.degree. C., 5 h, concentrated ammonia). In
those embodiments in which a base-sensitive group is attached to
the nucleic acids (e.g., TAMRA), the deprotection will preferably
use milder conditions (e.g., butylamine:water 1:3, 8 hours,
70.degree. C.). Deprotection under these conditions is facilitated
by the use of quick deprotect amidites (e.g., dC-acetyl,
dG-dmf).
Following cleavage from the support and deprotection, the nucleic
acid is purified by any method known in the art, including
chromatography, extraction and gel purification. In a preferred
embodiment, the nucleic acid is purified using HPLC. The
concentration and purity of the isolated nucleic acid is preferably
determined by measuring the optical density at 260 nm in a
spectrophotometer.
Peptide Probes
Peptides, proteins and peptide nucleic acids that are labeled with
a fluorophore and a PL of the invention can be used in both in vivo
and in vitro enzymatic assays.
Thus, in another aspect, the present invention provides a method
for determining whether a sample contains an enzyme. The method
comprises: (a) contacting the sample with a peptide construct; (b)
exciting the fluorophore; and (c) determining a fluorescence
property of the sample, wherein the presence of the enzyme in the
sample results in a change in the fluorescence property.
Peptide constructs useful in practicing the invention include those
with the following features: i) a fluorophore; ii) a PL of the
invention; and iii) a cleavage recognition site for the enzyme.
Moreover, the peptide construct is preferably of a length and
orientation and in a conformation allowing fluorescence energy
transfer between the fluorophore and the PL when the fluorophore is
excited.
When the probe is used to detect an enzyme, such as a degradative
enzyme (e.g., protease), and a degree of fluorescence resonance
energy transfer that is lower than an expected amount is observed,
this is generally indicative of the presence of an enzyme. The
degree of fluorescence resonance energy transfer in the sample can
be determined, for example, as a function of the amount of
fluorescence from the donor moiety, the amount of fluorescence from
the acceptor moiety, the ratio of the amount of fluorescence from
the donor moiety to the amount of fluorescence from the acceptor
moiety or the excitation state lifetime of the donor moiety.
The assay also is useful for determining the amount of enzyme in a
sample by determining the degree of fluorescence resonance energy
transfer at a first and second time after contact between the
enzyme and the tandem construct, and determining the difference in
the degree of fluorescence resonance energy transfer. The
difference in the degree of fluorescence resonance energy transfer
reflects the amount of enzyme in the sample.
The assay methods also can also be used to determine whether a
compound alters the activity of an enzyme, ie, screening assays.
Thus, in a further aspect, the invention provides methods of
determining the amount of activity of an enzyme in a sample from an
organism. The method includes: (a) contacting a sample comprising
the enzyme and the compound with a peptide construct comprising (b)
exciting the fluorophore; and (c) determining a fluorescence
property of the sample, wherein the activity of the enzyme in the
sample results in a change in the fluorescence property. Peptide
constructs useful in this aspect of the invention are substantially
similar to those described immediately above.
In a preferred embodiment, the amount of enzyme activity in the
sample is determined as a function of the degree of fluorescence
resonance energy transfer in the sample and the amount of activity
in the sample is compared with a standard activity for the same
amount of the enzyme. A difference between the amount of enzyme
activity in the sample and the standard activity indicates that the
compound alters the activity of the enzyme.
Representative enzymes with which the present invention can be
practiced include, for example, trypsin, enterokinase, HIV-1
protease, prohormone convertase, interleukin-1b-converting enzyme,
adenovirus endopeptidase, cytomegalovirus assemblin,
leishmanolysin, .beta.-secretase for amyloid precursor protein,
thrombin, renin, angiotensin-converting enzyme, cathepsin-D and a
kininogenase, and proteases in general.
Proteases play essential roles in many disease processes such as
Alzheimer's, hypertension, inflammation, apoptosis, and AIDS.
Compounds that block or enhance their activity have potential as
therapeutic agents. Because the normal substrates of peptidases are
linear peptides and because established procedures exist for making
non-peptidic analogs, compounds that affect the activity of
proteases are natural subjects of combinatorial chemistry.
Screening compounds produced by combinatorial chemistry requires
convenient enzymatic assays.
The most convenient assays for proteases are based on fluorescence
resonance energy transfer from a donor fluorophore to an acceptor
placed at opposite ends of a short peptide chain containing the
potential cleavage site (see, Knight C. G., Methods in Enzymol.
248:18-34 (1995)). Proteolysis separates the fluorophore and
acceptor, resulting in increased intensity in the emission of the
donor fluorophore. Existing protease assays use short peptide
substrates incorporating unnatural chromophoric amino acids,
assembled by solid phase peptide synthesis.
Assays of the invention are also useful for determining and
characterizing substrate cleavage sequences of proteases or for
identifying proteases, such as orphan proteases. In one embodiment
the method involves the replacement of a defined linker moiety
amino acid sequence with one that contains a randomized selection
of amino acids. A library of fluorescent PL-bearing probes, wherein
the fluorophore and the PL are linked by a randomized peptide
linker moiety can be generated using recombinant engineering
techniques or synthetic chemistry techniques. Screening the members
of the library can be accomplished by measuring a signal related to
cleavage, such as fluorescence energy transfer, after contacting
the cleavage enzyme with each of the library members of the tandem
fluorescent peptide construct. A degree of fluorescence resonance
energy transfer that is lower than an expected amount indicates the
presence of a linker sequence that is cleaved by the enzyme. The
degree of fluorescence resonance energy transfer in the sample can
be determined, for example, as a function of the amount of
fluorescence from the donor moiety, the amount of fluorescence from
the acceptor donor moiety, or the ratio of the amount of
fluorescence from the donor moiety to the amount of fluorescence
from the acceptor moiety or the excitation state lifetime of the
donor moiety.
In the tandem constructs of the invention, the donor and acceptor
moieties are connected through a linker moiety. The linker moiety,
preferably, includes a peptide moiety, but can be another organic
molecular moiety, as well. In a preferred embodiment, the linker
moiety includes a cleavage recognition site specific for an enzyme
or other cleavage agent of interest. A cleavage site in the linker
moiety is useful because when a tandem construct is mixed with the
cleavage agent, the linker is a substrate for cleavage by the
cleavage agent. Rupture of the linker moiety results in separation
of the fluorophore and the PL of the invention. The separation is
measurable as a change in FRET.
When the cleavage agent of interest is a protease, the linker can
comprise a peptide containing a cleavage recognition sequence for
the protease. A cleavage recognition sequence for a protease is a
specific amino acid sequence recognized by the protease during
proteolytic cleavage. Many protease cleavage sites are known in the
art, and these and other cleavage sites can be included in the
linker moiety. See, e.g., Matayoshi et al. Science 247: 954 (1990);
Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth.
Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615
(1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al.
Meth. Enzymol. 244: 412 (1994); Bouvier et al. Meth. Enzymol. 248:
614 (1995), Hardy et al., in AMYLOID PROTEIN PRECURSOR IN
DEVELOPMENT, AGING, AND ALZHEIMERS'S DISEASE, ed. Masters et al.
pp. 190-198(1994).
Solid Support Immobilized PL Analogues
The PLs of the invention can be immobilized on substantially any
polymer, biomolecule, and solid or semni-solid material having any
useful configuration. Moreover, any conjugate comprising one or
more PLs can be similarly immobilized. When the support is a solid
or semi-solid, examples of preferred types of supports for
immobilization of the nucleic acid probe include, but are not
limited to, controlled pore glass, glass plates, polystyrene,
avidin coated polystyrene beads, cellulose, nylon, acrylamide gel
and activated dextran. These solid supports are preferred because
of their chemical stability, ease of functionalization and
well-defined surface area. Solid supports such as, controlled pore
glass (CPG, 500 .ANG., 1000 .ANG.) and non-swelling high
cross-linked polystyrene (1000 .ANG.) are particularly
preferred.
According to the present invention, the surface of a solid support
is functionalized with a PL of the invention or a species including
a PL of the invention. For clarity of illustration, the following
discussion focuses on attaching a reactive PL to a solid support.
The following discussion is also broadly relevant to attaching a
species that includes within its structure a reactive PL to a solid
support, and the attachment of such species and reactive PL
analogues to other molecules and structures.
The PLs are preferably attached to a solid support by forming a
bond between a reactive group on the PL and a reactive group on the
surface of the solid support or a linker attached to the solid
support, thereby derivatizing the solid support with one or more PL
analogues. The bond between the solid support and the PL is
preferably a covalent bond, although ionic, dative and other such
bonds are useful as well. Reactive groups which can be used in
practicing the present invention are discussed in detail above and
include, for example, amines, hydroxyl groups, carboxylic acids,
carboxylic acid derivatives, alkenes, sulfhydryls, siloxanes,
etc.
A large number of solid supports appropriate for practicing the
present invention are available commercially and include, for
example, peptide synthesis resins, both with and without attached
amino acids and/or peptides (e.g., alkoxybenzyl alcohol resin,
arminomethyl resin, aminopolystyrene resin, benzhydrylamine resin,
etc. (Bachem)), functionalized controlled pore glass (BioSearch
Technologies, Inc.), ion exchange media (Aldrich), functionalized
membranes (e.g., --COOH membranes; Asahi Chemical Co., Asahi Glass
Co., and Tokuyama Soda Co.), and the like.
Moreover, for applications in which an appropriate solid support is
not commercially available, a wide variety of reaction types are
available for the functionalization of a solid support surface. For
example, supports constructed of a plastic such as polypropylene,
can be surface derivatized by chromic acid oxidation, and
subsequently converted to hydroxylated or aminomethylated surfaces.
The functionalized support is then reacted with a PL of
complementary reactivity, such as a PL active ester, acid chloride
or sulfonate ester, for example. Supports made from highly
crosslinked divinylbenzene can be surface derivatized by
chloromethylation and subsequent functional group manipulation.
Additionally, functionalized substrates can be made from etched,
reduced polytetrafluoroethylene.
When the support is constructed of a siliceous material such as
glass, the surface can be derivatized by reacting the surface
Si--OH, SiO--H, and/or Si--Si groups with a functionalizing
reagent.
In a preferred embodiment, wherein the substrates are made from
glass, the covalent bonding of the reactive group to the glass
surface is achieved by conversion of groups on the substrate's
surface by a silicon-modifying reagent such as:
where R.sup.a is an alkyl group, such as methyl or ethyl, R.sup.b
is a linking group between silicon and X.sup.a, and X.sup.2 is a
reactive group or a protected reactive group. Silane derivatives
having halogens or other leaving groups beside the displayed alkoxy
groups are also useful in the present invention.
In another preferred embodiment, the reagent used to functionalize
the solid support provides for more than one reactive group per
each reagent molecule. Using reagents, such as the compound below,
each reactive site on the substrate surface is, in essence,
"amplified" to two or more functional groups:
where R.sup.a is an alkyl group (e.g.,methyl, ethyl), R.sup.b is a
linking group between silicon and X.sup.a, X.sup.a is a reactive
group or a protected reactive group and n is an integer between 2
and 50, and more preferably between 2 and 20. The amplification of
a PL by its attachment to a silicon-containing substrate is
intended to be exemplary of the general concept of PL
amplification. This amplification strategy is equally applicable to
other aspects of the invention in which a PL analogue is attached
to another molecule or solid support.
A number of siloxane functionalizing reagents can be used, for
example: 1. Hydroxyalkyl siloxanes (Silylate surface, functionalize
with diborane, and H.sub.2 O.sub.2 to oxidize to the alcohol) a.
allyl trichlorosilane.fwdarw..fwdarw.3-hydroxypropyl b.
7-oct-1-enyl trichlorchlorosilane.fwdarw..fwdarw.8-hydroxyoctyl 2.
Diol (dihydroxyalkyl) siloxanes (silylate surface and hydrolyze to
diol) a. (glycidyl
trimethoxysilane.fwdarw..fwdarw.(2,3-dihydroxypropyloxy)propyl 3.
Aminoalkyl siloxanes (amines requiring no intermediate
functionalizing step) a. 3-aminopropyl
trimethoxysilane.fwdarw.aminopropyl 4. Dimeric secondary aminoalkyl
siloxanes a.
bis(3-trimethoxysilylpropyl)amine.fwdarw.bis(silyloxylpropyl)amine.
It will be apparent to those of skill in the art that an array of
similarly useful functionalizing chemistries is available when
support components other than siloxanes are used. Thus, for example
alkyl thiols, functionalized as discussed above in the context of
siloxane-modifying reagents, can be attached to metal films and
subsequently reacted with a PL to produce the immobilized compound
of the invention.
R groups of use for R.sup.b in the above described embodiments of
the present invention include, but are not limited to, alkyl,
substituted alkyl, aryl, arylalkyl, substituted aryl, substituted
arylalkyl, acyl, halogen, hydroxy, amino, alkylamino, acylamino,
alkoxy, acyloxy, aryloxy, aryloxyalkyl, mercapto, saturated cyclic
hydrocarbon, unsaturated cyclic hydrocarbon, heteroaryl,
heteroarylalkyl, substituted heteroaryl, substituted
heteroarylalkyl, heterocyclic, substituted heterocyclic and
heterocyclicalkyl groups and combinations thereof.
Nucleic Acid Capture Probes
In one embodiment, an immobilized nucleic acid comprising a PL is
used as a capture probe. The nucleic acid probe can be attached
directly to a solid support, for example by attachment of the 3'-
or 5'-terminal nucleotide of the probe to the solid support. More
preferably, however, the probe is attached to the solid support by
a linker (i.e., spacer arm, supra). The linker serves to distance
the probe from the solid support. The linker is most preferably
from about 5 to about 30 atoms in length, more preferably from
about 10 to about 50 atoms in length.
In yet another preferred embodiment, the solid support is also used
as the synthesis support in preparing the probe. The length and
chemical stability of the linker between the solid support and the
first 3'-unit of nucleic acid play an important role in efficient
synthesis and hybridization of support bound nucleic acids. The
linker arm should be sufficiently long so that a high yield
(>97%) can be achieved during automated synthesis. The required
length of the linker will depend on the particular solid support
used. For example, a six atom linker is generally sufficient to
achieve a >97% yield during automated synthesis of nucleic acids
when high cross-linked polystyrene is used as the solid support.
The linker arm is preferably at least 20 atoms long in order to
attain a high yield (>97%) during automated synthesis when CPG
is used as the solid support.
Hybridization of a probe immobilized on a solid support generally
requires that the probe be separated from the solid support by at
least 30 atoms, more preferably at least 50 atoms. In order to
achieve this separation, the linker generally includes a spacer
positioned between the liner and the 3'-terminus. For nucleic acid
synthesis, the linker arm is usually attached to the 3'-OH of the
3'-terminus by an ester linkage which can be cleaved with basic
reagents to free the nucleic acid from the solid support.
A wide variety of linkers are known in the art, which may be used
to attach the nucleic acid probe to the solid support. The linker
may be formed of any compound, which does not significantly
interfere with the hybridization of the target sequence to the
probe attached to the solid support. The linker may be formed of,
for example, a homopolymeric nucleic acid, which can be readily
added on to the linker by automated synthesis. Alternatively,
polymers such as functionalized polyethylene glycol can be used as
the linker. Such polymers are presently preferred over
homopolymeric nucleic acids because they do not significantly
interfere with the hybridization of probe to the target nucleic
acid. Polyethylene glycol is particularly preferred because it is
commercially available, soluble in both organic and aqueous media,
easy to functionalize, and completely stable under nucleic acid
synthesis and post-synthesis conditions.
The linkages between the solid support, the linker and the probe
are preferably not cleaved during synthesis or removal of base
protecting groups under basic conditions at high temperature. These
linkages can, however, be selected from groups that are cleavable
under a variety of conditions. Examples of presently preferred
linkages include carbamate, ester and amide linkages.
Acrylamide-Immobilized Probes
In another preferred embodiment, a species is within a matrix, such
as an acrylamide matrix and the species bears a PL, or the presence
of the immobilized species is ascertained using a probe bearing a
PL. In a preferred embodiment, the immobilization is accomplished
in conjunction with the "acrydite" process invented and
commercialized by Mosaic Technologies (Cambridge, Mass., see,
Rehman et al., Nucleic Acids Research, 27: 649-655 (1999)). The
acrydite method allows immobilization of alkene labeled capture
probes within a polymerized polyacrylamide network. When target
mixes are run past the immobilized probe band under electrophoresis
conditions, the target nucleic acid is captured substantially
quantitatively. However, detection of this event currently requires
a second probe. In one embodiment, probes bearing a PL, and/or a
fluorphore, are immobilized in an acrylamide matrix and
subsequently contacted with the target mix. By using fluorescent
probes as capture probes, signals from target mixes can be directly
detected in real time.
Microarrays
The invention also provides microarrays including immobilized PLs
and compounds functionalized with PLs. Moreover, the invention
provides methods of interrogating microarrays using probes that are
functionalized with PLs. The immobilized species and the probes are
selected from substantially any type of molecule, including, but
not limited to, small molecules, peptides, enzymes nucleic acids
and the like.
Nucleic acid microairays consisting of a multitude of immobilized
nucleic acids are revolutionary tools for the generation of genomic
information, see, Debouck et al., in supplement to Nature Genetics,
21:48-50 (1999). The discussion that follows focuses on the use of
PLs in conjunction with nucleic acid microarrays. This focus is
intended to be illustrative and does not limit the scope of
materials with which this aspect of the present invention can be
practiced.
Thus, in another preferred embodiment, the compounds of the present
invention are utilized in a microarray format. The PLs, or species
bearing PLs can themselves be components of a microarray or,
alternatively they can be utilized as a tool to screen components
of a microarray.
Thus, in a preferred embodiment, the present invention provides a
method of screening a microarray. The method includes contacting
the members of the microarray with a PL-bearing probe and
interrogating the microarray for regions of fluorescence. The
fluorescent regions are indicative of the presence of an
interaction between the PL-bearing probe and a microarray
component. In another version of this method, the microarray is
interrogated for regions in which fluorescence is quenched, again
indicating the presence of an interaction between the PL-bearing
probe and a component of the microarray.
In another preferred embodiment, the array comprises immobilized
PL-bearing FET probes as the interrogating species. In this
embodiment, the probe "turns on" when hybridized to its target.
Such arrays are easily prepared and read, and can be designed to
give quantitative data. Arrays comprising PL-bearing probes are
valuable tools for expression analysis and clinical genomic
screening.
In another preferred embodiment, the immobilized PL-bearing probe
is not a FET probe. A microarray based on such as format can be
used to probe for the presence of interactions between an analyte
and the immobilized probe by, for example, observing the quenching
of analyte fluorescence upon interaction between the probe and
analyte.
In a further preferred embodiment, the microarrays comprise n
probes that comprise identical or different nucleic acid sequences.
Alternatively, the microarray can comprise a mixture of n probes
comprising groups of identical and different nucleic acid sequences
identical nucleic acid sequences). In a preferred embodiment, n is
a number from 2 to 100, more preferably, from 10 to 1,000, and more
preferably from 100 to 10,000. In a still further preferred
embodiment, the n probes are patterned on a substrate as n distinct
locations in a manner that allows the identity of each of the n
locations to be ascertained.
In yet another preferred embodiment, the invention also provides a
method for preparing a microarray of n PL-bearing probes. The
method includes attaching PL-bearing probes to selected regions of
a substrate. A variety of methods are currently available for
making arrays of biological macromolecules, such as arrays nucleic
acid molecules. The following discussion focuses on the assembly of
a microatray of PL-bearing probes, this focus is for reasons of
brevity and is intended to be illustrative and not limiting.
One method for making ordered arrays of PL-bearing probes on a
substrate is a "dot blot" approach. In this method, a vacuum
manifold transfers a plurality, e.g., 96, aqueous samples of probes
from 3 millimeter diameter wells to a substrate. The probe is
immobilized on the porous membrane by baking the membrane or
exposing it to UV radiation. A common variant of this procedure is
a "slot-blot" method in which the wells have highly-elongated oval
shapes.
Another technique employed for making ordered arrays of probes uses
an array of pins dipped into the wells, e.g., the 96 wells of a
microtiter plate, for transferring an array of samples to a
substrate, such as a porous membrane. One array includes pins that
are designed to spot a membrane in a staggered fashion, for
creating an array of 9216 spots in a 22.times.22 cm area. See,
Lehrach, et al., HYBRIDIZATION FINGERPRINTING IN GENOME MAPPING AND
SEQUENCING, GENOME ANALYSIS, Vol. 1, Davies et al, Eds., Cold
Springs Harbor Press, pp. 39-81 (1990).
An alternate method of creating ordered arrays of probes is
analogous to that described by Pirrung et al. (U.S. Pat. No.
5,143,854, issued 1992), and also by Fodor et al., (Science, 251:
767-773 (1991)). This method involves synthesizing different probes
at different discrete regions of a particle or other substrate.
This method is preferably used with relatively short probe
molecules, e.g., less than 20 bases. A related method has been
described by Southern et al. (Genomics, 13: 1008-1017 (1992)).
Khrapko, et al., DNA Sequence, 1: 375-388 (1991) describes a method
of making an nucleic acid matrix by spotting DNA onto a thin layer
of polyacrylamide. The spotting is done manually with a
micropipette.
The substrate can also be patterned using techniques such as
photolithography (Kleinfield et al., J. Neurosci. 8:4098-120
(1998)), photoetching, chemical etching and microcontact printing
(Kumar et al., Langmuir 10:1498-511 (1994)). Other techniques for
forming patterns on a substrate will be readily apparent to those
of skill in the art.
The size and complexity of the pattern on the substrate is limited
only by the resolution of the technique utilized and the purpose
for which the pattern is intended. For example, using microcontact
printing, features as small as 200 nm are layered onto a substrate.
See, Xia, Y., J. Am. Chem. Soc. 117:3274-75 (1995). Similarly,
using photolithography, patterns with features as small as 1 .mu.m
are produced. See, Hickman et al., J. Vac. Sci. Technol. 12:607-16
(1994). Patterns which are useful in the present invention include
those which include features such as wells, enclosures, partitions,
recesses, inlets, outlets, channels, troughs, diffraction gratings
and the like.
In a presently preferred embodiment, the patterning is used to
produce a substrate having a plurality of adjacent wells,
indentations or holes to contain the probes. In general, each of
these substrate features is isolated from the other wells by a
raised wall or partition and the wells do not fluidically
communicate. Thus, a particle, or other substance, placed in a
particular well remains substantially confined to that well. In
another preferred embodiment, the patterning allows the creation of
channels through the device whereby an analyte or other substance
can enter and/or exit the device.
In another embodiment, the probes are immobilized by "printing"
them directly onto a substrate or, alternatively, a "lift off"
technique can be utilized. In the lift off technique, a patterned
resist is laid onto the substrate, an organic layer is laid down in
those areas not covered by the resist and the resist is
subsequently removed. Resists appropriate for use with the
substrates of the present invention are known to those of skill in
the art. See, for example, Kleinfield et al., J. Neurosci.
8:4098-120 (1998). Following removal of the photoresist, a second
CAP, having a structure different from the first probe can be
bonded to the substrate on those areas initially covered by the
resist. Using this technique, substrates with patterns of probes
having different characteristics can be produced. Similar substrate
configurations are accessible through microprinting a layer with
the desired characteristics directly onto the substrate. See,
Mrkish et al. Ann. Rev. Biophys. Biomol. Struct. 25:55-78
(1996).
Spacer Groups
As used herein, the term "spacer group," refers to constituents of
PL-bearing probes. The spacer group links donor and/or acceptor
moieties and other groups to the nucleic acid, peptide or other
polymeric component of the probe. The spacer groups can be
hydrophilic (e.g., tetraethylene glycol, hexaethylene glycol,
polyethylene glycol) or they can be hydrophobic (e.g., hexane,
decane, etc.).
In a preferred embodiment, the immobilized construct includes a
spacer between the solid support reactive group and the PL
analogue. The linker is preferably selected from C.sub.6 -C.sub.30
alkyl groups, C.sub.6 -C.sub.30 substituted alkyl groups, polyols,
polyethers (e.g., poly(ethyleneglycol)), polyamines, polyamino
acids, polysaccharides and combinations thereof.
In certain embodiments, it is advantageous to have a moiety of the
probe attached to the polymeric component by a group that provides
flexibility and distance from the polymeric component. Using such
spacer groups, the properties of the moiety adjacent to the
polymeric component is modulated. Properties that are usefully
controlled include, for example, hydrophobicity, hydrophilicity,
surface-activity, the distance of the donor and/or PL moiety from
the nucleic acid and the distance of the donor from the PL.
In an exemplary embodiment, the spacer serves to distance the PL
from a nucleic acid. Spacers with this characteristic have several
uses. For example, a PL held too closely to the nucleic acid may
not interact with the donor group, or it may interact with too low
of an affinity. When a PL is itself sterically demanding, the
interaction leading to quenching can be undesirably weakened, or it
may not occur at all, due to a sterically-induced hindering of the
approach of the two components.
When the construct comprising the PL is immobilized by attachment
to, for example, a solid support, the construct can also include a
spacer moiety between the reactive group of the solid support and
the PL analogue, or other probe component bound to the solid
support.
In yet a further embodiment, a spacer group used in the probes of
the invention is provided with a group that can be cleaved to
release a bound moiety, such as a PL, fluorophore, minor groove
binder, intercalating moiety, and the like from the polymeric
component. Many cleaveable groups are known in the art. See, for
example, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983);
Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et
al., J. Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J.
Biochem., 155: 141-147 (1986); Park et al., J. Biol. Chem., 261:
205-210 (1986); Browning et al., J. Immunol., 143: 1859-1867
(1989). Moreover a broad range of cleavable, bifunctional (both
homo- and hetero-bifunctional) spacer arms are commercially
available from suppliers such as Pierce.
An exemplary embodiment utilizing spacer groups is set forth in
Formulae VII and VIII, above. In these formulae, R.sup.b is either
stable or it can be cleaved by chemical or photochemical reactions.
For example, R.sup.b groups comprising ester or disulfide bonds can
be cleaved by hydrolysis and reduction, respectively. Also within
the scope of the present invention is the use of R.sup.b groups
which are cleaved by light such as, for example, nitrobenzyl
derivatives, phenacyl groups, benzoin esters, etc. Other such
cleaveable groups are well-known to those of skill in the art.
Kits
In another aspect, the present invention provides kits containing
one or more of the PLs or PL-bearing compositions of the invention.
In one embodiment, a kit will include a reactive PL derivative and
directions for attaching this derivative to another molecule. In
another embodiment, the kit include a PL-labeled nucleic acid that
optionally is also labeled with a second fluorophore or quencher
and directions for using this nucleic acid in one or more assay
formats. Other formats for kits will be apparent to those of skill
in the art and are within the scope of the present invention.
The invention provides kits for practicing the methods noted above.
The kits can include any of the compositions noted above, and
optionally further include additional components such as
instructions to practice the methods, one or more containers or
compartments (e.g., to hold the assay components, nucleic acids,
antibodies, inhibitors or the like), a robotic armature for mixing
kit components or the like.
The invention also provides integrated systems for performing the
methods disclosed herein. For example, in the performing assays, in
one embodiment, the delivery of individual compounds or compound
components is accomplished by means of a robotic armature which
transfers fluid from a source to a destination, a controller which
controls the robotic armature, a label detector, a data storage
unit which records label detection, and an assay component such as
a microtiter dish comprising a well. When a labeled compound is
used, it is detected by means of the label detector.
A number of robotic fluid transfer systems are available, or can
easily be made from existing components. For example, a Zymate XP
(Zymark Corporation; Hopkinton, Mass.) automated robot using a
Microlab 2200 (Hamilton; Reno, Nev.) pipetting station can be used
to transfer parallel samples to 96 well microtiter plates to set up
several parallel simultaneous ligation reactions.
Optical Amplification
Optical signals are important for transmitting information.
However, when an optical signal is transmitted through an optical
fiber, attenuation will always occur to a certain extent, such that
it is necessary to amplify the signal after a certain distance
(typically in the order of about 50-100 km). Conventionally, for
that purpose an electronic amplifier is used. At the amplifier
station, the optical signal must then be converted into an
electrical signal, which is amplified in an electronic amplifer,
after which the amplified electrical signal is converted back into
an optical signal. This involves not only the disadvantage that an
amplifier station has a rather complicated structure with rather a
large number of parts, among which optical/electrical converters
and electrical/optical converters, but this also implies that the
bandwidth and bit-rate of the overall system is limited by the
electronic components. Therefore, optical fiber amplifiers have
recently been developed, i.e. amplifiers which amplify the optical
signal directly and do not need a conversion into an electrical
signal. Such devices are disclosed in, for example, Yan et al.,
U.S. Pat. No. 5,982,973, issued Nov. 9, 1999; Kleinerman, U.S. Pat.
No. 5,928,222, issued Jul. 27, 1999; Desurvire, Physics Today,
January 1994, 20-27; Sloof et al., J. Appl. Phys. 83: 497
(1998).
Thus, in another embodiment, the present invention provides a
substrate for the transmission and amplification of light, said
substrate comprising a compound of the invention. The compound of
the invention can be incorporated into the substrate in any manner
known in the art, including, but not limited to, covalent
attachment, coating, doping, and the like.
The substrate can include any material useful for a particular
application, including, but not limited to, glass, organic
polymers, inorganic polymers and combinations thereof.
Also provided is a method for amplifying light transmitted by the
substrate derivatized with a compound of the invention, as
described above. The method comprises transmitting light through
such a substrate, thereby amplifying the light.
The substrates and methods of the invention can be used in fiber
optic devices, sensors (see, for example, Kopelman et al., U.S.
Pat. No. 5,627,922; and Pinkel et al., U.S. Pat. No. 5,690,894),
fiber optic "refrigerators" and the like.
Medical Applications
The compounds of the invention can also be used to treat malignant
tumors via photodynamic therapy (PDT). Additionally, the complexes
of the invention be used in vivo and in vitro as chelating agents
for: (1) certain paramagnetic metal ions to achieve higher contrast
in magnetic resonance imaging (MRI); and (2) radioactive metal ions
for tumor imaging in single-photon-emission tomography (SPECT) or
position emission tomography (PET) and/or in radioisotope-mediated
radiation therapy. Thus, appropriately radiolabeled phthalamide
chelates can be imaged noninvasively in nuclear medicine employing
SPECT or PET. See, for example, Margerum et al., U.S. Pat. No.
6,010,681; and Woodburn et al., U.S. Pat. No. 6,022,526.
Separations
In another preferred embodiment, the specificity of the compounds
of the invention for particular ions in solution is exploited to
separate those ions from other solutes, including ions for which a
compound of the invention has a lower affinity or specificity. In a
preferred embodiment, the PLs are used to separate one lanthanide
ion from another. Many examples of ion selective or ion specific
chelating agents are known in the art. See, for example, Izatt, et
al. Synthesis of Macrocycles Wiley-Interscience, New York, 1987;
and Martell et al., Determination and Use of Stability Constants,
2.sup.nd Ed., VCH Publishers, New York, 1992.
The materials, methods and devices of the present invention are
further illustrated by the examples which follow. These examples
are offered to illustrate, but not to limit the claimed
invention.
EXAMPLES
Example 1 illustrates the synthesis of exemplary ligands of the
invention and the formation of their metal complexes. The synthetic
scheme for the compounds of Example 1 is set forth in FIG. 1.
Example 2 details the x-ray structure determination of the
compounds of the invention. Molecular models based on the x-ray
crystallographic data are set forth in FIG. 4 and FIG. 5.
Example 3 details the spectrophotometric titration of exemplary
compounds of the invention.
Example 4 details the determination of the quantum yield of
exemplary compounds of the invention.
Example 5 illustrates high resolution luminescence measurements of
exemplary compounds of the invention.
Example 6 illustrates the synthesis of the ligand H22IAM, its
analogues and metal chelates. The synthetic scheme for the
compounds of Example 6 is set forth in FIG. 8.
Example 7 illustrates the synthesis of exemplary bicapped ligands
of the invention and their metal chelates.
Example 8 sets forth studies on the photophysical and stability
properties of complexes with the ligand H22IAM in aqueous
solution.
Example 9 describes studies on the photophysical and stability
properties of complexes with the ligands H22IAM and bicapped H22IAM
in DMSO.
Example 10 illustrates the synthesis of the ligand
H22IAM-mono-(N-5aminopentylsuccinamic acid), 18. This ligand has
been designed to connect the luminescent lanthanide complex to an
immunoreactive species such as antibodies. One of the four
2-hydroxyisophthalamide groups is substituted with a linker
terminated with a carboxylic group. The synthetic scheme is set
forth in FIG. 9.
Example 11 illustrates the synthesis of the Ligand
H22tetra(6-amino-1-hexaneamido)IAM, 23. This ligand has been
designed to connect the luminescent lanthanide complex to a
biomolecule, such as an immunoreactive species (e.g., antibodies).
The four 2-hydroxyisophthalamide groups are substituted with a
linker terminated with primary amines. The synthesis of 23 is set
forth in FIG. 10.
Example 12 illustrates the synthesis of compounds of the invention
having backbones of variable length. The synthetic scheme is set
forth in FIG. 21.
Example 1
Example 1 illustrates the synthesis of exemplary ligands of the
invention and the formation of their metal complexes. The synthetic
scheme for the compounds of Example 1 is set forth in FIG. 1. 1.1
Materials and Methods
Unless otherwise noted, starting materials were obtained from
commercial suppliers and used without further purification. Flash
column chromatography was performed using Merck silica gel 40-70
mesh. Microanalyses were performed by the Microanalytical Services
Laboratory, College of Chemistry, University of California,
Berkeley. Mass spectra were recorded at the Mass Spectrometry
Laboratory, College of Chemistry, University of California,
Berkeley. .sup.1 H and .sup.13 C NMR spectra were recorded on an
AMX 300 or AMX 400 Bruker superconducting Fourier transform
spectrometer or on a DRX 500 Brucker superconducting digital
spectrometer. Infrared spectra were measured using a Nicolet Magna
IR 550 Fourier transform spectrometer. The UV/Visible spectra were
recorded on a double-beam Perkin-Elmer Lambda 9 UV-Visible
spectrophotometer. 1.2 Snthesis of 2-Methoxyisophthalic
acid-bis(2-mercaptothiazolide) (1)
2-Methoxyisophthalic acid (0.02 mol), 2-mercaptothiazoline (0.04
mol), and 4-dimethylaminopyridine (20 mg) were dissolved in 150 mL
of CH.sub.2 Cl.sub.2 under a nitrogen atmosphere.
1,3-Dicyclohexylcarbodiimide (0.04 mol) was added to the reaction
mixture which gradually became yellow in color. After stirring for
5 hours the reaction mixture was filtered and the filtrate
evaporated to dryness to afford a yellow oil. Recrystallization
from hot ethyl acetate gave a bright yellow microcrystalline solid.
Yield: 55%. IR (film from CDCl.sub.3) .nu.1229, 1684, 2955
cm.sup.-1. .sup.1 H NMR (300 MHz, CDCl.sub.3, 25.degree. C.):
.delta.3.42 (t, J=7.3 Hz, 2H, CH.sub.2), 3.89 (s, 3H, CH.sub.3),
4.60 (t, J=7.3 Hz, 2H, CH.sub.2), 7.13 (t, J=7.7 Hz, 1H, ArH), 7.43
(d, J=7.6 Hz, 2H, ArH). .sup.13 C NMR (400 MHz, CDCl.sub.3,
25.degree. C.): .delta.29.2, 55.6, 62.9, 123.1, 128.1, 131.9,
154.7, 167.1, 200.8. Anal. Calcd (Found) for C.sub.15 H.sub.14
N.sub.2 O.sub.3 S.sub.4.0.25 H.sub.2 O: C, 44.70 (44.75); H, 3.63
(3.53); N, 6.95 (6.82). 1.3 Synthesis of Me.sub.3
(TRENIAM)-tris(2-mercaptothiazolide) (2)
Tris(2-aminoethyl)amine (TREN) (1.7 mmol) dissolved in 100 mL of
CH.sub.2 Cl.sub.2 was added dropwise to 1 (15.1 mmol) dissolved in
600 mL of CH.sub.2 Cl.sub.2. The reaction mixture was evaporated to
dryness to give a yellow oil. The oil was purified by flash silica
column chromatography with 0-4% MeOH/CH.sub.2 Cl.sub.2. The solvent
was evaporated to give the product as a yellow foam. Yield: 70%. IR
(film from CDCl.sub.3) .nu.1635, 2939, 3386 cm.sup.-1. .sup.1 H NMR
(500 MHz, CDCl.sub.3, 25.degree. C.): .delta.2.83 (s, 6H,
CH.sub.2), 3.42 (br t, 6H, CH.sub.2), 3.84 (s, 9H, CH.sub.3), 4.63
(br t, 6H, CH.sub.2), 7.18 (t, J=7.7 Hz, 3H, ArH), 7.39 (d, J=5.7
Hz, 3H, ArH), 7.73 (br t, 3H, NH), 8.01 (d, J=6.0 Hz, 3H, ArH).
.sup.13 C NMR (500 MHz, CDCl.sub.3, 25.degree. C.): .delta.29.2,
38.2, 53.6, 55.7, 63.2, 124.4, 127.1, 129.1, 132.2, 134.1, 155.6,
165.0, 167.3, 201.4. Anal. Calcd (Found) for C.sub.42 H.sub.45
N.sub.7 O.sub.9 S.sub.6 : C, 51.26 (51.26); H, 4.61 (4.71); N, 9.96
(10.09). 1.4 Synthesis of Me.sub.3 (bicappedTRENSAM) (3)
2 (7.0 mmol) dissolved in 400 mL of CHCl.sub.3 and TREN (6.9 mmol)
dissolved in 400 mL of CHCl.sub.3 were added using laboratory pumps
to a 5-L round-bottom flask filled with 2800 mL of CHCl.sub.3 and
fitted with a mechanical overhead stirrer. The mixture was heated
during the addition to .about.40.degree. C. under a nitrogen
atmosphere. After 24 hrs, the reaction mixture was purified on a
silica column, eluted with 0-10% MeOH/CH.sub.2 Cl.sub.2 gradient.
The solvent was evaporated to give the product as a colorless
glass. Yield: 46%. IR (film from CDCl.sub.3) .nu.1522, 1652, 2939
cm.sup.-1. .sup.1 H NMR (500 MHz, CDCl.sub.3, 25.degree. C.):
.delta.3.45 (s, 24H, CH.sub.2), 3.47 (s, 9H, CH.sub.3), 7.15 (t,
J=7.7 3H, ArH), 7.54 (br t, 6H, NH), 7.80 (d, J=7.7 Hz, 6H, ArH).
.sup.13 C NMR (500 MHz, CDCl.sub.3, 25.degree. C.): .delta.39.4,
56.2, 62.5, 124.2, 128.2, 133.1, 155.7, 166.1. (+)-FABMS: m/z: 773
[M.sup.+ +H]. Anal. Calcd. (Found) for C.sub.39 H.sub.48
N.sub.8.sub.9.2MeOH: C, 58.84 (58.68); H, 6.74 (6.73); N, 13.39
(13.19). 1.5 Synthesis of H.sub.3 (bicapedTRENSAM).2HBr (4)
3 (0.3 mmol) was dissolved in 30 mL of dry, degassed CH.sub.2
Cl.sub.2. To the cooled solution was added BBr.sub.3 (42.3 mmol)
via syringe under a nitrogen atmosphere. After stirring for
.about.36 hrs the solution was evaporated to dryness to get a pale
orange solid. The solid was slowly quenched with MeOH and added to
100 mL of boiling water. The solution was boiled for 3.5 hrs and
then allowed to cool, precipitating a white solid. The solid was
collected by filtration and oven dried. Yield: 80%. .sup.1 H NMR
(300 MHz, DMSO-d.sub.6, 25.degree. C.): .delta.3.67 (s, 24H,
CH.sub.2), 6.97 (t, J=7.9 Hz, 3H, ArH), 8.00 (d, J=7.8 Hz, 6H,
ArH), 9.10 (br s, 6H, NH). .sup.13 C NMR (500 MHz, DMSO-d.sub.6,
25.degree. C.): .delta.36.2, 57.1, 117.6, 118.9, 134.1, 159.6,
169.0. (+)-FABMS: m/z: 731 [M.sup.+ +H]. Anal. Calcd. (Found) for
C.sub.36 H.sub.44 N.sub.8 O.sub.9 Br.sub.2.3H.sub.2 O: C, 45.68
(45.81); H, 5.32 (5.47); N, 11.84 (11.66). 1.6 Synthesis of
[Eu(bicappedTRENSAM).sub.2 ]Br(DMF)(H.sub.2 O).sub.10
4 (0.50 mmol) was dissolved in 5 mL of DMSO and 10 mL of water to
which the lanthanide salt (0.24 mmol, Ln(NO.sub.3).sub.3 or
LnCl.sub.3) was added. The suspension was heated to reflux until
all the solids dissolved (20 min), after which an excess of
pyridine (0.5 mL) was added. The mixture was refluxed for 5 hrs and
then cooled to room temperature. The solution was evaporated to
dryness and the residue was suspended in iPrOH, sonicated, and
filtered. Yield: 80%. (+)-FABMS: m/z: 1612 [M.sup.+ +2H]. Anal.
Calcd. (Found) for C.sub.75 H.sub.109 N.sub.17 O.sub.29 BrEu: C,
46.13 (46.32); H, 5.63 (5.65); N, 12.19 (12.24). 1.7 Synthesis of
[Tb(bicappedTRENSAM).sub.2 ].sup.+ 5
The same procedure was used as described in the synthesis of the
Eu.sup.3+ complex. Yield: 80%. (+)-FABMS: m/z: 1619 [M.sup.+ +H].
1.8 Results
The macrobicycle ligands were synthesized under high dilution
conditions by reaction of the appropriate poly(thiazolide)
intermediate with one equivalent of polyamine (Karpishin ET AL., J.
Am. Chem. Soc. 115: 182 (1993)). This provided the protected ligand
as a colorless foams which were deprotected using BBr.sub.3. When
illuminated at 254 and 365 nm in aqueous solution, the ligand is
highly luminescent, emitting blue light.
Synthesis of the metal complexes is performed by suspending the
ligand in aqueous solution (or a DMSO/water mixture) followed by
addition of the appropriate lanthanide salt. After refluxing for
about 30 minutes, an excess of base is added (pyridine or
triethylamine) and the reaction is continued for several more
hours. The formation of the metal complexes can be monitored by
irradiation of the aqueous reaction mixtures with a UV lamp (254
and 365 nm). When illuminated, the reaction mixture of the
Th.sup.3+ complex emits bright green light, while that of the
Eu.sup.3+ complex emits red light. Both colors are readily visible
with the naked eye. The luminescence of both the Eu.sup.3+ and
Tb.sup.3+ complexes remains very bright after isolation and drying
of the compounds. The replacement of the blue fluorescence of the
ligand by green or red luminescence when Tb.sup.3+ or Eu.sup.3+ are
added to the solution suggests that there is efficient energy
transfer from the ligand to the metal ion. As coordinated water is
known to be very efficient at quenching the excited states of
Eu.sup.3+ (.sup.5 D.sub.0) and Tb.sup.3+ (.sup.5 D.sub.4), the
strong luminescence in aqueous solvent suggests that the ligands
offer good protection against the binding of water in the first
coordination sphere of the lanthanide cation (Bunzli, J.-C. G.
Luminescent Probes; Bunzli, J.-C. G. and Choppin, G. R., Ed.;
Elsevier: Amsterdam, 1989, pp 245).
Example 2
Example 2 details the x-ray structure determination of exemplary
compounds of the invention. Molecular models based on the x-ray
crystallographic data are set forth in FIG. 4 and FIG. 5. 2.1 X-ray
Data Collection, Structure Solutions, and Refinement
All X-ray structure data sets were collected on a Siemens SMART
Area Detector diffractometer (SMART, Area-Detector Software
Package; Siemens Industrial Automation, Inc.: Madison, 1994).
Crystals were mounted on quartz capillaries in Paratone oil and
were cooled in a nitrogen stream on the diffractometer. Peak
integrations were performed using Siemens SAINT software package
(SAINT, SAX Area-Detector Integration Program v. 4.024; Siemens
Industrial Automation, Inc.: Madison, 1994). Space group
determinations were done by the software XPREP. The structures were
solved by direct methods and refined using the SHELXTL software
package (PC version, SHELXTL, Crystal Structure Analysis
Determination Package; Siemens Industrial Automation, Inc.:
Madison, 1994). All hydrogen atoms were fixed at calculated
positions and their thermal parameters refined isotropically; all
non-hydrogen atoms were refined anisotropically. 2.2 Results
The synthesis of the lanthanide complexes using an excess of ligand
allowed for the isolation of exclusively the ML.sub.2 complex.
Crystals of [Eu(bicapped TRENSAM).sub.2 ].sup.+ suitable for X-ray
diffraction were grown from diffusion of acetone into a wet DMF
solution of the complex. The structure shows the wrapping of two
ligands of bicapped TRENSAM around the hemispheres of the metal
center (FIG. 4). The ligands approach the metal in an orthogonal
fashion, about 90.degree. offset from one another. The Eu.sup.3+
metal center is eight coordinate using two of the three binding
units from each of two different cryptates. The coordination
polyhedron around Eu.sup.3+ can be described as a slightly
distorted square-antiprism with each face of the antiprism made up
of one of the macrobicycles (FIG. 5). See, Kepert, D. L. Inorganic
Stereochemistry; Springer-Verlag: Berlin, 1982. The third arm of
each macrobicycle does not coordinate, in a fashion similar to that
seen in some metal-oxo macrobicyclic complexes like MoO.sub.2
[bicapped TRENCAM] (Albrecht, M.; Franklin, S. J.; Raymond, K. N.
Inorg. Chem. 1994, 33, 5785). The non-coordinating isophthalamide
ligands participate in an intramolecular .pi.-.pi. stacking
interaction with one of the bound chelators, which may contribute
to protecting the metal center from solvent coordination. This
non-coordinating chelate may also be responsible for one of the two
main bands that appear at low energy on the UV-Vis spectra of this
complex (FIG. 2). The space filling model indicates that the
assembly of the two ligands around the metal ion provides very
effective protection against deactivation of the excited state of
the lanthanide metal ions by solvent molecules. The structure also
shows that the complex is stabilized in part due to the strong
intramolecular hydrogen bond formed between the carboxylamide
proton and the deprotonated 2-hydroxyl oxygen of each
isophthalamide chelate (Cohen, S. M.; Raymond, K. N. manuscript in
preparation 1998)
Example 3
Example 3 details the spectrophotometric titration of exemplary
compounds of the invention. 3.1 Spectrophotometric Titrations
Batch titration samples were prepared in Millipore purified water
with MES buffer, 0.1 M KCl, adjusted to pH=5.78 with KOH. The
samples were incubated at 37.degree. C. for 15 hours before
measurement to ensure thermodynamic equilibrium had been reached.
The ligand concentration was 2.32.times.10.sup.-5 M for all samples
and the EuCl.sub.3 was titrated from 0 to 2.2 equivalents. The
spectra were recorded on a double-beam Perkin-Elmer Lambda 9
UV-Visible spectrophotometer in 1.0 cm quartz Suprasil cell. The
samples were kept at a constant temperature of 25.0.+-.0.2.degree.
C. using a Neslab RTE-111 water bath. The treatment of the data was
performed with the using the software package SPECFIT
2.10..sup.86.1 3.2 Results
A batch titration of bicapped TRENSAM with Eu.sup.3+ was performed
in order to evaluate the solution behavior and stability of these
complexes. The concentration of the ligand was kept constant and
increasing amounts of lanthanide ion were added to a series of
samples. The experimental spectra for this titration are shown in
FIG. 2A. The spectra show the emergence of new bands with
increasing metal concentration. From the experimental spectra it is
readily apparent that two different types of metal complexes are
formed, one with a single absoption band and one with a double
absorption band. Using the program Specfit factor analysis revealed
three absorbing species present during the titration (Gampp, H.;
Maeder, M.; Meyer, C. J.; Zuberbuler, A. D. Talanta 1986, 33, 943).
The spectrum for the free ligand was measured independently and the
calculated spectrum for the metal species (ML, and ML.sub.2) were
deconvoluted as shown in FIG. 2B. A comparison of the calculated
and observed spectra for the ML.sub.2 complex (which was
synthesized independently in the presence of excess ligand) further
confirms the accuracy of the mathematical treatment of the
experimental data (FIG. 3). The stability constants of Ln[bicapped
TRENSAM].sub.n (log.beta..sub.11 and log.beta..sub.12 of Eu.sup.3+
with bicapped TRENSAM) are 7.8 for the ML complex and 14.4 for the
ML.sub.2 complex, respectively. These stabilities are sufficient
for use in fluoroimmunoassay systems (Bunzli, J.-C. G. Luminescent
Probes; Bunzli, J.-C. G. and Choppin, G. R., Ed.; Elsevier:
Amsterdam, 1989, pp 219).
Example 4
Example 4 details the determination of the quantum yield of
exemplary compounds of the invention. 4.1 Quantum Yield
Determination
The quantum yield of sample solutions containing bicapped TRENSAM,
and Tb.sup.3+ [bicappedTRENSAM].sub.n were determined in water
(Millipore purified) relative to quinine sulfate in 0.05 M H.sub.2
SO.sub.4 (refractive index: 1.338, absolute quantum yield: 0.54).
See, Meech, S. R.; Phillips, D. C. J. Photochem. 1983, 23, 193. The
quantum yields of both Eu.sup.3+ [bicappedTRENSAM].sub.n complexes
were determined with the solutions from the spectrophotometric
titration (vide supra) (stoichimetry ratio M:L=0.5 for the
Eu.sup.3+ [bicappedTRENSAM].sub.2 complex and M:L=1.0 for the
Eu.sup.3+ [bicappedTRENSAM] complex). Measurements under ligand
excitation conditions were performed on a Spex FluoroMax
spectrofluorimeter with an excitation beam-centered 0.100 cm quartz
Suprasil luminescence cell. The relative quantum yield (Q.sub.x
/Q.sub.r) was calculated using Equation 1.
The subscript r refers to the reference and x to the samples. The
remaining terms in the equation are defined as follows: A is the
absorbance at the excitation wavelength, I is the intensity of the
excitation light at the same wavelength, n is the refractive index,
and D is the integrated luminescence intensity. Measured samples
were prepared at concentrations between 1.times.10.sup.-3
-1.times.10.sup.-4 M.
Example 5
Example 5 illustrates high resolution luminescence measurements of
exemplary compounds of the invention. 5.1 High-resolution
Luminescence Measurements
High resolution luminescence spectra were measured on a previously
described instrumental setup (Bunzli, J.-C. G.; Milicic-Tang, A.
Inorg. Chim. Acta 1996, 252, 221). Samples were measured as finely
powdered samples (La, Eu, Tb)and as monocrystals (Eu). Bidistilled
water was used for aqueous; the pH of the solutions was around 6.5.
Luminescence spectra were corrected for the instrumental function
but not excitation spectra. 5.2 Results
The free ligand bicapped TRENSAM, as well as the Eu.sup.3+ and
Tb.sup.3+ metal complexes formed with this ligand, are extremely
fluorescent in water. FIG. 6 shows the emission spectra in aqueous
solution of [Eu(bicappedTRENSAM).sub.2 ].sup.+,
[Tb(bicappedTRENSAM).sub.2 ].sup.+, and free bicapped TRENSAM
excited at 320, 338, and 302 nm, respectively. The emission
spectrum of the free ligand bicapped TRENSAM reveals an intense
broad fluorescence band centered around 398 mn which gives rise to
the blue emitted light. However, upon addition of the metal ion the
spectra of the complexes are comprised of only intense narrow lines
originating from metal centered f.fwdarw.f transitions. In
addition, the broad ligand fluorescence band vanishes completely
upon metal complexation, which suggests an efficient conversion of
the UV light absorbed by the ligand into the visible red
(Eu.sup.3+) or green (Tb.sup.3+) luminescence of the lanthanide
ions.
High resolution, solid state measurements were performed on a
crystalline sample of the [Eu(bicappedTRENSAM).sub.2 ].sup.+
complex. Analysis of the .sup.5 D.sub.0.fwdarw..sup.7 F.sub.J
transitions in terms of crystal field splitting shows the .sup.7
F.sub.1 level split into three, almost equally spaced components,
pointing to a low site symmetry. .sup.7 F.sub.2 is split into three
components and .sup.7 F.sub.4 into 5-6 components, which, combined
with the very low intensity of the .sup.5 D.sub.0.fwdarw..sup.7
F.sub.0 points to a D.sub.2 site symmetry (number of allowed
transitions: 0, 3, 3 and 6 for J=0, 1, 2, and 4). The luminescence
data therefore confirm the X-ray analysis of the coordination
polyhedron around Eu.sup.3+, described as a distorted square
antiprism (D.sub.4d.fwdarw.D.sub.4.fwdarw.D.sub.2). The emission
spectrum of a 10.sup.-4 M aqueous solution presents essentially the
same features as the emission spectrum of the solid sample at 295 K
and the .sup.5 D.sub.0.rarw..sup.7 F.sub.0 excitation spectrum
shows a single, broad band centered at 17 243 cm.sup.-1 (fwhh=11.9
cm.sup.-1), pointing to an Eu.sup.III environment in solution
similar to the one in the solid state.
The quantum yields of the Eu(bicappedTRENSAM).sub.n complexes have
been determined in water with 0.1 M KCl, 0.05 MES buffer, adjusted
to pH 5.78 with KOH. Both Eu(bicappedTRENSAM).sub.n complexes are
highly luminescent with quantum yields for the EuL and EuL.sub.2
complexes of approximately 1% and 10%, respectively. The quantum
yield measured in water for Tb(bicappedTRENSAM).sub.2 was
determined to be at least 36%. The quantum yield of the free ligand
in water was measured to be about 9%.
The quantum yields of these complexes are much higher than those
found for polypyridine complexes used in recently developed
single-step, luminescent assay systems, where the quantum yield in
water is 2% (Mathis, G. Clin. Chem. 1993, 39, 1953; Mathis, G.
Clin. Chem. 1995, 41, 1391). In addition, bicapped TRENSAM is
unusual in that it forms highly luminescent complexes with both
Tb.sup.3+ and Eu.sup.3+. In most ligand systems that have been
investigated, strong luminescence is only observed with either
Tb.sup.3+ or Eu.sup.3+, but not both. The origin of why this ligand
produces such luminescent complexes with both metal ions will
require further spectroscopic measurements. These experiments will
determine the overall electronic structure of the complex and in
particular the positions of the donating energy levels of the
ligand and of the accepting metal orbitals (Petoud, S.; Bunzli,
J.-C. G.; Schenk, K. J.; Piguet, C. Inorg. Chem. 1997, 36,
1345).
Example 6
Example 6 illustrates the synthesis of the ligand H22IAM, its
analogues and metal chelates. The synthetic scheme for the
compounds of Example 6 is set forth in FIG. 8. 6.1 Synthesis of
tetrakis(thiazoline)Me.sub.4 H22IAM, 7
Tetrakis(2-aminoethyl)ethylenediamine (H22) (2.8 mmol) was
dissolved in 150 mL of CH.sub.2 Cl.sub.2 was added dropwise to 1
(82.9 mmol) dissolved in 600 mL of CH.sub.2 Cl.sub.2. The reaction
mixture was evaporated to dryness to get a yellow oil. The oil was
purified by flash silica column chromatography (0-15% MeOH in
CH.sub.2 Cl.sub.2 gradient). The solvent was evaporated to give the
product 7 as a yellow foam. Yield: 83.8%. IR (film from CH.sub.2
Cl.sub.2) .nu.1522, 1653, 2942 cm.sup.-1. .sup.1 H NMR (500 MHz,
CDCl.sub.3, 25.degree. C.) .delta.2.67 (s, 4H, CH.sub.2), 2.71 (t,
J=6.2 Hz, 8H, CH.sub.2), 3.39 (br t, 8H, CH.sub.2), 3.48 (s, J=5.8
Hz, 8H, CH.sub.2), 3.76 (s, 12H, OCH.sub.3), 4.59 (t, J=7.2 Hz, 8H,
CH.sub.2), 7.14 (t, J=7.7 Hz, 4H, ArH), 7.35 (d, J=5.8 Hz, 4H,
ArH), 7.79 (br t, 4H, NH), 7.97 (d, J=6.0 Hz, 4H, ArH). .sup.13 C
NMR (500 MHz, CDCl.sub.3, 25.degree. C.) .delta.29.1, 37.9, 50.6,
53.5, 55.7, 63.1, 124.3, 127.2, 129.1, 132.0, 133.9, 155.6, 164.9,
167.3, 201.4. Calcd. (Found) for C.sub.58 H.sub.64 N.sub.10
O.sub.12 S.sub.8.2CH.sub.2 Cl.sub.2 : C, 47.43 (47.45); H, 4.51
(4.52); N, 9.22 (9.54). 6.2 Synthesis of Me.sub.4 H22IAM, 8
7 (1.1 mmol) was dissolved in 10 mL of CH.sub.2 Cl.sub.2. To this
solution was added 1.5 mL of aqueous methylamine (40% by weight),
followed by vigorous shaking of the biphasic mixture. Within 30 sec
all the yellow color was gone. The mixture was evaporated to
dryness and the remaining residue purified by flash silica column
chromatography (0-15% MeOH in CH.sub.2 Cl.sub.2 gradient). Removal
of solvent and oven drying gave 8 as a white foam. Yield: 84.1%. IR
(film from CDCl.sub.3) .nu.1539, 1652, 3296 cm.sup.-1. .sup.1 H NMR
(500 MHz, CDCl.sub.3, 25.degree. C.) .delta.2.74 (br m, 12H,
CH.sub.2), 2.87 (d, J=4.5 Hz, 12H, NCH.sub.3), 3.40 (br d, 8H,
CH.sub.2), 3.70 (s, 12H, OCH.sub.3), 7.02 (t, J=7.5 Hz, 4H, ArH),
7.50 (br d, 4H, NH), 7.62 (d, J=7.5 Hz, 4H, ArH), 7.72 (d, J=8.0
Hz, 4H, ArH), 7.83 (br t, 4H, NH). .sup.13 C NMR (500 MHz,
CDCl.sub.3, 25.degree. C.) .delta.26.7, 37.7, 51.7, 53.3, 63.1,
124.5, 127.4, 128.7, 133.1, 133.5, 155.6, 165.5, 166.2. Calcd.
(Found) for C.sub.50 H.sub.64 N.sub.10 O.sub.12.2H.sub.2 O: C,
58.13 (58.30); H, 6.63 (6.59); N, 13.56 (13.75). 63 Synthesis of
H.sub.4 H22IAM.2HBr, 9
8 (0.9 mmol) was dissolved in 40 mL of dry, degassed CH.sub.2
Cl.sub.2. The solution was cooled in an ice bath and BBr.sub.3
(48.0 mmol) was added via syringe under a nitrogen atmosphere. The
pale yellow slurry was stirred for 48 hrs, after which the solution
was slowly quenched with MeOH. The mixture was diluted with water
(total volume 100 mL) and boiled until all the yellow color was
gone. The resulting colorless solution was boiled to a volume of 50
mL and then cooled, affording a white solid. The product was
collected by filtration and oven dried. Yield: 74%. .sup.1 H NMR
(500 MHz, DMSO-d.sub.6, 25.degree. C.) .delta.2.81 (d, J=4.5 Hz,
12H, NCH.sub.3), 3.48 (br s, 8H, CH.sub.2), 3.76 (br s, 12H,
CH.sub.2), 6.92 (t, J=7.5 Hz, 4H, ArH), 7.97 (d, J=7.6 Hz, 4H,
ArH), 8.02 (d, J=7.6 Hz, 4H, ArH), 8.87 (br s, 4H, NH), 8.97 (br s,
4H, NH), 10.09 (br s, 4H, OH). .sup.13 C NMR (500 MHz,
DMSO-d.sub.6, 25.degree. C.) .delta.26.5, 35.0, 52.9, 116.6, 118.5,
119.8, 132.0, 134.6, 160.2, 166.9, 169.4. Calcd. (Found) for
C.sub.46 H.sub.58 N.sub.10 O.sub.12 Br.sub.2.5H.sub.2 O: C, 46.32
(46.34); H, 5.75 (5.70); N, 11.74 (11.66). (+)-FABMS: m/z: 941
[M.sup.+ +H]. 6.4 Synthesis of [Eu(H22IAM)].sup.+, 10
8 (0.09 mmol) and EuCl.sub.2.7H.sub.2 O (0.09 mmol, 99.999%) are
suspended in a mixture of 5 mL H.sub.2 O and 1.5 mL MeOH and
heated. At reflux temperature the solution is clear and 1.4 mL of
pyridine are added. A white solid started to precipitate. The
reacting mixture is red when irradiated by UV light at 354 nm.
After 20 hours of reaction, the solvent is reduced and the mixture
is cooled at 4.degree. C. After filtration of the white solid and
drying in a vacuum oven, 95 mg of product are collected. Yield:
81.7%. Calcd. (Found) for EuC.sub.46 H.sub.54 N.sub.10
O.sub.12.1Br.4H.sub.2 O: C, 44.45 (44.67); H, 5.03 (5.01); N, 11.27
(11.09). 6.5 Synthesis of [Tb(H22IAM)].sup.+, 11
8 (0.18 mmol) and Tb(NO.sub.3).sub.3.6H.sub.2 O (0.18 mmol,
99.999%) are suspended in a mixture of 11.0 mL H.sub.2 O and 2.5 mL
MeOH. The procedure was similar to what was described for the
synthesis of [Eu(H22IAM)].sup.+. Calcd. (Found) for TbC.sub.46
H.sub.52 N.sub.10 O.sub.12. 1Br.5H.sub.2 O: C, 43.66 (43.70); H,
4.94 (4.97); N, 11.08 (11.20). 6.6 Synthesis of [La(H22IAM].sup.+,
12
8 (0.05 mmol) and LaCl.sub.2.7H.sub.2 O (0.05 mmol, 99.999%) are
suspended in a mixture of 2.5 mL H.sub.2 O and 1 mL MeOH. The
procedure was similar to what was described for the synthesis of
[Eu(H22IAM)].sup.+. 46 mg of white product was collected after
isolation and drying. Yield: 74.8%. Calcd. (Found) for LaC.sub.46
H.sub.54 N.sub.10 O.sub.12.1Br.4H.sub.2 O: C, 44.92 (44.91); H,
5.08 (5.09); N, 11.39 (11.15).
Example 7
Example 7 illustrates the synthesis of exemplary bicapped ligands
of the invention and their metal chelates. 7.1 Synthesis of
Me.sub.4 Bicaped H22IAM, 13
7 (2.3 mmol) dissolved in 400 mL of CHCl.sub.3 and H22 amine (2.2
mmol) dissolved in 400 mL of CHCl.sub.3 were added using laboratory
pumps to a 5-L round-bottom flask filled with 2400 mL of CHCl.sub.3
and fitted with a mechanical overhead stirrer and a small reflux
condenser. The reaction mixture was heated to 55.degree. C. and
kept under a nitrogen atmosphere. After .about.31 hrs, the mixture
was evaporated to dryness and the remaining residue purified by
flash silica column chromatography (0-20% MeOH in CHCl.sub.3
gradient). The solvent was evaporated to give the product as an
pale amber foam. This compound exists as two conformational isomers
as indicated by .sup.1 H and .sup.13 C NMR (this is also found with
protected bicapped H22TAM). Only the spectra for the major isomer
are reported. Yield: 44.0%. .sup.1 H NMR (500 MHz, CDCl.sub.3,
25.degree. C.) .delta.2.69 (br s, 32H, CH.sub.2), 2.80 (s, 8H,
CH.sub.2), 3.42 (br s, 12H, OCH.sub.3), 7.00 (br t, 4H, ArH), 7.53
(br s, 8H, NH, 7.65 (d, J=7.5 Hz, 8H, ArH). .sup.13 C NMR (500 MHz,
CDCl.sub.3, 25.degree. C.) .delta.37.9, 51.2, 53.2, 62.8, 124.4,
128.1, 132.8, 155.4, 165.7. Calcd. (Found) for C.sub.56 H.sub.72
N.sub.12 O.sub.12.1MeOH.0.5CH.sub.2 Cl.sub.2 : C, 58.54 (58.18); H,
6.58 (6.81); N, 14.25 (14.16). (+)-FABMS: m/z: 1106 [M.sup.30 +H],
1144 [M.sup.+ +K]. 7.2 Synthesis of H.sub.4 Bicapped H22IAM.4HBr,
14
13 (0.74 mmol) was dissolved in 40 mL of dry, degassed CH.sub.2
Cl.sub.2. The solution was cooled in an ice bath and BBr.sub.3
(48.0 mmol) was added via syringe under N.sub.2(g). The pale yellow
slurry was stirred for 24 hrs, after which the solution was slowly
quenched with MeOH. The mixture was diluted with MeOH (total volume
100 mL) and boiled until most of the yellow color was gone. The
resulting slurry was boiled to a volume of 50 mL and then cooled to
affording a beige solid. The product was collected by filtration
and oven dried. The ligand undergoes some undetermined
conformational dynamics at room temperature which broaden both the
.sup.1 H and .sup.13 C NMR spectrum. Yield: 91%. .sup.1 H NMR (500
MHz, DMSO-d.sub.6, 90.degree. C.) , .delta.3.45/3.77 (br s, 40H,
CH.sub.2), 6.70 (br t, 4H, ArH), 7.85 (br d, 8H, ArH), 8.64 (br s,
8H, NH). .sup.13 C NMR (500 MHz, DMSO-d.sub.6, 25.degree. C.)
.delta.16.8, 37.0 (br), 52.6 (br), 109.4, 118.2, 133.8 (br), 159.9,
167.8 (br). Calcd. (Found) for C.sub.52 H.sub.68 N.sub.12 O.sub.12
Br.sub.4.4H.sub.2 O: C, 43.23 (42.11); H, 5.30 (5.44); N, 11.63
(11.26). (+)-FABMS: m/z: 1050 [M.sup.+ +H]. 7.3 Synthesis of
Tb[bicapped]H22IAM, 15
14 (0.50 mmol) was dissolved in 5 mL of DMSO and 10 mL of water to
which Tb(NO.sub.3).sub.3.6H.sub.2 O (0.24 mmol, 99.999%) was added.
The suspension was heated to reflux (30 min), after which an excess
of triethylamine (0.5 mL) was added. The mixture was refluxed for 5
hrs and then cooled to room temperature. The solution was
evaporated to remove the water and the remaining solution was
diluted with THF to precipitate a white solid. The solid was
collected by filtration and oven dried. Yield: 80%. (+)-FABMS: m/z:
1203 [M.sup.+ +H].
Example 8
Example 8 sets forth studies on the photophysical and stability
properties of complexes with the ligand H22IAM in aqueous solution.
8.1 Conditions for the Aqueous Measurements at Low
Concentration
To avoid hydrolysis of the lanthanide, it is desirable to use a
phosphate buffer when measurements of solution of lanthanide
complex are performed below the concentration of 10.sup.-7 M. The
reported measurements were measured in solution buffered at pH=7.4
(physiological pH) with 0.01 M potassium phosphate buffer (8.02 mL
of K.sub.2 HPO.sub.4 +1.98 mL of KH.sub.2 PO.sub.4 diluted to 1000
mL). The phosphate buffer is commonly used in fluoroimmunoassay
technology. The pH domain of this buffer is compatible with the
measurement conditions of time-resolved fluoroimmunoassays as well
as for the conjugation with antibodies.
The strong blue luminescence of the ligand H22IAM can be detected
with the naked eye. The maximum of the emission is located around
425 nm. The excitation spectra of the compound reveals to
excitation bands at 240 and 340 nm, the latter being more efficient
for the excitation of the complex. The maximum of the broad
absorption band is located at 340 nm.
FIG. 13 is a normalized excitation (dotted line, .lambda..sub.an
=417 nm) and emission (full line, .lambda..sub.ex =350 nm) spectra
of the ligand H22IAM .about.10.sup.-6 M in Millipore water.
FIG. 14 is a UV/vis spectrum of [Tb(H22IAM)].sup.+
8.2.multidot.10.sup.-7 M in Millipore water, 1.000 cm cell.
No significant emission band arising from the ligand can be
observed in the luminescence spectra of the complexes
[Tb(H22IAM)].sup.+ and [Eu(H22IAM)].sup.+. This observation
indicates that an efficient ligand to metal energy transfer takes
place in both Eu.sup.3+ and Tb.sup.3+ complexes.
FIG. 15 is a normalized emission spectra of [Tb(H22IAM)].sup.+ and
[Eu(H22IAM)].sup.+ in Millipore water. [Tb(H22IAM)].sup.+
8.2.multidot.10.sup.-7 M, .lambda..sub.ex =354 nm;
[Eu(H22IAM)].sup.+.about.10.sup.-6 M, .lambda..sub.ex =350 nm. 8.2
Quantum Yields
Table 3 displays the absolute quantum yields of [Tb(H22IAM)].sup.+,
at 8.2.multidot.10.sup.-7 M in aerated Millipore water, measured by
comparison with quinine sulfate in H.sub.2 SO.sub.4 0.05 M
(absolute quantum yield: 0.5460). Samples and reference were
measured at the same absorption and at the same .lambda..sub.ex.
The solutions were prepared 48 hours before the experiment. The
solution A was protected from light and the solution B was exposed
to the ambient light during the 48 hour delay.
TABLE 3 Solution .lambda..sub.exc /nm Q.sub.Abs A 354 0.6045 A 318
0.6051 B 354 0.5557 B 318 0.5111
The high value of quantum yield obtained with the complex
[Tb(H22IAM)].sup.+ indicates a very efficacy of the energy transfer
occurring from the ligand to the Tb.sup.3+ cation. 8.3
Lifetimes
A preliminary measurement of the lifetime of the Tb.sup.3+ in a
8.2.multidot.10.sup.-7 M solution of [Tb(H22IAM)].sup.+ was
performed using a laser excitation and a fast acquisition system. A
unique lifetime value of 2.56 ms was obtained under ligand
excitation (.lambda..sub.ex =352 nm, .lambda..sub.an =545 nm). This
result indicates that the Tb.sup.3+ cation is well protected by the
ligand H22IAM against water coordination, preventing non-radiative
deactivation. 8.4 Stability in Water
An estimate of the stability of the [Tb(H22IAM)].sup.+ complex can
be obtained by performing a dilution experiment. The luminescence
of a molecule is mostly dependent on its quantum yield an to its
extinction coefficient. As the lanthanide cations have extremely
low extinction coefficient, a ligand coordinated to the lanthanide
must be able to act as an "antenna". An "antenna" is a molecule
able to absorb a high quantity of UV light and to transfer the
resulting energy to the lanthanide cation. This implies that the
luminescence of the lanthanide cation can only be observed if the
complex is still formed in solution and can be used to estimate the
stability of the luminescent lanthanide complex. A stock solution
of [Tb(H22IAM)].sup.+ in phosphate buffer was successively diluted
and the emission spectra was recorded. The results are set forth in
FIG. 16, which contains emission spectra of the complex
[Tb(H22IAM)].sup.+ in phosphate buffer 0.01 M at various
concentration. .lambda..sub.ex =347 mn.
These results indicate that the luminescence of the complexes can
be detected in a 10.sup.-15 M solution, indicating a very high
thermodynamic stability of this complex. This behavior can not be
the results of a kinetic inertness since the complexes were
synthesized in water.
Example 9
Example 9 describes studies on the photophysical and stability
properties of complexes with the ligands H22IAM and bicapped H22IAM
in DMSO. 9.1 Quantum Yields
In order to obtain quantitative values on the energy transfer and
quenching solvent effect, the relative quantum yields of
[Tb(H22IAM)].sup.+ and [Tb(bicapped H22IAM)].sup.+ were determined
in DMSO using [Tb(bicapped TRENSAM).sub.2 ].sup.+ as a reference.
The results are reported in Table 3, which displays the relative
quantum yields of [Tb(H22IAM)].sup.+ 9.98.multidot.10.sup.-5 M and
[Tb(bicappedH22IAM)].sup.+ 9.19.multidot.10.sup.-5 M determined in
DMSO by using [Tb(bicapped TRENSAM).sub.2 ].sup.+
1.071.multidot.10.sup.-4 M as a reference. Sample and reference
were measured at the same absorption and at the same
.lambda..sub.ex except a where the sample was excited at 378 nm and
the reference at 372 nm.
TABLE 3 Compound .lambda..sub.exc /nm A(.lambda..sub.exc) Q.sub.Rel
Tb(bicappedTRENSAM).sub.2 335 0.2316 1.0000 365 0.0926 1.0000 372
0.0462 1.0000 Tb(H22IAM) 335 0.2401 4.6672 365 0.1644 3.6027 372
0.0937 3.2873 378 0.0462 3.4055.sup.a Tb(bicappedH22IAM) 365 0.0794
1.7757 372 0.0456 1.3610
The comparison of the quantum yields of [Ln(H22IAM)].sup.+ and
[Ln(bicappedH22IAM)].sup.+ revealed better luminescence properties
for the more stable complex formed with [Ln(H22IAM)].sup.+. One
other interesting result was obtained from the emission spectrum of
the [Tb(bicappedTRENSAM).sub.2 ].sup.+ complex. In addition to the
typical transition arising from the .sup.5 D.sub.4 levels of the
Tb.sup.3+, a broad band arising from the ligands' electronics
levels can be observed at higher energy. This observation reveals
the ligand.fwdarw.Ln energy transfer is not totally efficient in
DMSO unlike in water where no emission arising from the ligand was
observed. 9.2 Stability in DMSO
In order to estimate the stability of [Tb(H22IAM)].sup.+ and
[Tb(bicappedH22IAM)].sup.+ in DMSO, the emission spectra of these
compounds were measured at different concentrations.
The results of these studies are displayed in FIG. 17, which
displays normalized emission spectra of [Tb(H22IAM)].sup.+ at
various concentrations. .lambda..sub.ex, =335 nm (9.98.10.sup.-5 M)
and .lambda..sub.ex =347 nm for all others concentrations. The
signal arising from the Tb.sup.3+ could be discriminated and
amplified by a time-resolved measurement, and FIG. 18, which
displays normalized emission spectra of [Tb(bicappedH22IAM)].sup.+
at various concentrations. .lambda..sub.ex =365 nm
(9.19.multidot.10.sup.-5 M) and .lambda..sub.ex =351 nm for all
other concentrations. The signal arising from the Tb.sup.3+ could
be discriminated and amplified by a time-resolved measurement.
Example 10
Example 10 illustrates the synthesis of the ligand
H22IAM-mono-(N-5-aminopentylsuccinamic acid), 18. This ligand has
been designed to connect the luminescent lanthanide complex to an
immunoreactive species such as antibodies. One of the four
2-hydroxyisophthalamide groups is substituted with a linker
terminated with a carboxylic group. The synthetic scheme is set
forth in FIG. 9. 10.1 Synthesis of 10-Amino-4-oxo-5-aza-decanoic
acid, 16
To a solution of 1,5-pentanediamine (2.5 g, 24 mmol) in dry
methanol (20 mL), a solution of succinic acid anhydride (2 g, 20
mmol) in dry methylene chloride (20 mL) was added dropwise in 2
hrs, the product was precipitated as white solid and collected by
filtration. Yield 80%. .sup.1 H NMR (500 MHz, D.sub.2 O, 25.degree.
C.) .delta.1.280 (quint., J=7.5, 2H, CH.sub.2), 1.449 (quint.,
J=7.5, 2H, CH.sub.2), 1.577 (quint., J=7.5, 2H, CH.sub.2) 2.337 (s,
4H, CH.sub.2), 2.886 (t, J=7.5, 2H, CH.sub.2), 3.072 (t, J=7.5, 2H,
CH.sub.2). .sup.13 C NMR (500 MHz, D.sub.2 O, 25.degree. C.)
.delta.22.73, 26.22, 27.66, 32.34, 33.13, 38.76, 39.37, 175.72,
180.90. 10.2 Me.sub.4 H22IAM-mono-(N-5-aminopentyl)succinamic acid
derivative, 18
To a solution of Me.sub.4 H22IAMtetrakis(thiazolide) (7, FIG. 8).
(5.4 g, 4 mmol) in CH.sub.2 Cl.sub.2 (500 mL), 16 (0.2 g, 1 mmol)
in CH.sub.2 Cl.sub.2 (200 mL) was added dropwise over 24 hrs and
the mixture was stirred for another 24 hrs, then methylamine (0.1
mL, 40% aqueous solution) was added. The characteristic yellow
color of the thiazolide disappeared; the solution was evaporated to
dryness and the appropriate fractions were separated on a gradient
flash silica gel chromatography column (5-10% CH.sub.3 OH in
CH.sub.2 Cl.sub.2) to give 0.59 g (51%) pure product as white foam.
(+)-FABMS: m/Z: 1154.7 [MH.sup.+ ]. .sup.1 H NMR (500 MHz,
CDCl.sub.3, 25.degree. C.) .delta.1.246 (br s, 2H, CH.sub.2), 1.371
(br s, 2H, CH.sub.2), 1.469 (br s, 2H, CH.sub.2), 2.263 (br s, 2H,
CH.sub.2), 2.355 (br s, 2H, CH.sub.2), 2.737 (br s, 12H,
NCH.sub.2), 2.858 (s, 4H, CH.sub.2), 3.015 (br s, 2H, CH.sub.2),
3.275 (br s, 2H, CH.sub.2), 3.439 (s, 8H, NCH.sub.2), 3.694 (s,
12H, OCH.sub.3), 6.930 (s, 1H, NH), 7.020(t, J=7.5, 4H, ArH),
7.5-7.6 (m, 8H, 4NH+4ArH), 7.722 (d, 4H, J=7.0, ArH), 7.94-8.0 (m,
4H, NH). 13C NMR (500 MHz, CDCl.sub.3, 25.degree. C.),
.delta.=21.50, 23.8, 26.57, 28.63, 30.66, 31.24, 37.19, 38.90,
39.43, 44.91, 50.88, 52.95, 62.99, 63.03, 124.31, 127.34, 127.40,
128.41, 128.45, 128.66, 132.91, 133.39, 155.46, 155.52, 165.62,
166.12, 173.02, 175.10, 176.51. 10.3
H22IAM-mono-(N-5-aminopentyl-succinamic acid) derivative, 19
18 (0.58 g, 0.5 mmol) was dissolved in dry degassed CH.sub.2
Cl.sub.2 (20 mL). The solution was cooled in an ice bath and
BBr.sub.3 (1 mL, 11.4 mmol) was added via syringe under nitrogen.
The resulted pale yellow slurry was stirred for 48 hrs, after which
the volatile product was removed under vacuum and the residue
quenched with methanol (30 mL). The methanol solution was diluted
with water (40 mL) and boiled until a colorless transparent
solution was obtained; its volume was reduced to 10 mL. The
solution was cooled, and a white solid started to precipitate, 46
mg of product was collected by filtration and vacuum dried. The
filtrate of this solution was collected by centrifugation and
additional 213 mg of solid was obtained after drying. Yield: 40%.
(+)-FABMS: m/Z: 1097.7. .sup.1 H NMR (500 MHz, DMSO-d.sub.6, 25
.degree. C.) .delta.1.24-1.60 (m, 6H, CH.sub.2), 2.812 (m, 4H,
CH.sub.2), 3.476 (br s, 12H, NCH.sub.2), 3.702 (br s, 8H,
NCH.sub.2), 6.923 (t, J=7.7, 4H, ArH), 7.684 (br s, 1H, NH),
7.95-8.04 (m, 8H, ArH), 8.818 (br s, 4H, NH), 8.943 (br s, 4H, NH).
.sup.1 H NMR (500 MHz, D.sub.2 O, 25.degree. C.) .delta.1.297
(quint, J=7.2, 2H, CH.sub.2), 1.444 (quint, J=7.2, 2H, CH.sub.2),
1.579 (quint, J=7.2, 2H, CH.sub.2), 2.664 (s, 4H, CH.sub.2), 2.889
(t, J=7.2, 2H, CH.sub.2), 3.099 (t, J=7.2, 2H, CH.sub.2), 3.437 (br
s, 8H, NCH.sub.2) 3.653 (br s, 12H, NCH.sub.2), 6.5-6.7 (m, 4H,
ArH), 7.3-7.5 (m, 8H, ArH). Calcd. (Found) for C.sub.54 H.sub.69
N.sub.11 O.sub.15 (HBr).sub.2.9H.sub.2 O: C, 45.16 (45.14); H, 5.49
(6.25); N, 10.46 (10.73). 10.4
Tb[H22IAM-mono-(N-5-aminopentyl-succinamic acid) derivative],
19
18 (0.042 mmol) and Tb(NO.sub.3).sub.3.6H.sub.2 O (0.042 mmol,
99.999%) were suspended in 30 mL of MeOH. The suspension was heated
until reflux and became clear. The solvent was reduced to 7 ml and
a large excess of pyridine was added. A white solid that strongly
emits a green color under UV irradiation (354 nm) precipitated
instantaneously. The suspension was maintained under reflux for 15
hours. After cooling of the solution to 4.degree. C. and filtration
of the product, 46 mg of solid were collected after being dried in
vacuum oven. Yield: 63%. Calcd. (Found) for TbC.sub.54 H.sub.68
N.sub.11 O.sub.15 Br.sub.2 (NO.sub.3).7H.sub.2 O: C, 40.09 (39.95);
H, 5.11 (4.76); N, 10.39 (10.42). 10.5
Eu[H22IAM-mono-N-5-aminopentyl-succinamic acid derivative], 20
18 (0.014 mmol) was suspended in 12 mL of H.sub.2 O containing
EuCl.sub.3.7H.sub.2 O (0.014 mmol, 99.999%). The suspension was
heated until reflux. When the solution was clear, an excess of
pyridine was added (30 drops). A white precipitate appeared
instantaneously and was redissolved upon further pyridine addition.
After 15 hours of reflux, the solvent was removed and the white
solid was redissolved in MeOH. The solid was obtained by addition
of pyridine and a strong red emission was observed upon UV
irradiation (354 nm). 11.4 mg of compound was collected after
filtration and drying. Yield: 53%. Calcd (Found) for EuC.sub.54
H.sub.68 N.sub.11 O.sub.15 Br.sub.2 Cl.5H.sub.2 O: C, 41.89
(41.55); H, 5.08 (5.07); N, 9.95 (10.24).
Example 11
Example 11 illustrates the synthesis of the Ligand
H22tetra(6-amino-1-hexaneamido)IAM, 23. This ligand has been
designed to connect the luminescent lanthanide complex to an
immunoreactive species such as antibodies. The four
2-hydroxyisophthalamide groups are substituted with a linker
terminated with primary amines. The synthesis of 23 is set forth in
FIG. 10. 11.1 Synthesis of 6-(Z-amino)-1-hexylamine, 21
A solution of 1,6-hexanediamine (0.050 mmol) in CH.sub.2 Cl.sub.2
(50 mL) was neutralized with 1.0 equivalent of hydrochloric acid.
To this half neutralized amine solution, a solution of Z-thiazolide
in CH.sub.2 Cl.sub.2 (50 mL) was added slowly in 8 hrs with
stirring. The resulted solution was washed with 2 M KOH solution
(100 mL) and then evaporated to dryness. The residue was then
purified by chromatography (3-15% CH.sub.3 OH in CH.sub.2 Cl.sub.2
gradient), 6-(Z-amino)-1-hexaneamine was obtained as a white
semisolid. Yield: 52%. .sup.1 HNMR (500 MHz, CDCl.sub.3, 25.degree.
C.) .delta.1.3193 (br s, 4H, CH.sub.2), 1.4255 (br d, 2H,
CH.sub.2), 1.4875 (br d,), 2.6671 (t, 2H, J=7.0, 2H, CH.sub.2),
3.1765 (q, 2H, J=6.5, 2H, CH.sub.2), 4.8532 (br s, 1H, NH), 5.0827
(s, 2H, OCH.sub.2), 7.28-7.38 (m, 5H, ArH). 11.2 Me.sub.4
H22tetra(6-Z-amino)-1-hexaneamido)IAM, 22
To a solution of Me4H22tetrakis(thiazolide)IAM (7, FIG. 8) (0.8
mmol) in CH.sub.2 Cl.sub.2 (50 mL), 21 (4 mmol) was added, the
mixture was stirred until the characteristic yellow color of the
tetrathiazolide disappears. The reaction mixture was evaporated to
dryness, the appropriate fractions were collected from of a
gradient flash silica gel column (2-7% CH.sub.3 OH in CH.sub.2
Cl.sub.2) and were evaporated to dryness to gave 0.89 g (59%) pure
product as white foam. (+)-FABMS: m/Z: 1874.9 [MH.sup.+ ]. .sup.1 H
NMR (500 MHz, CDCl.sub.3, 25.degree. C.) .delta.1.333 (br s, 16H,
CH.sub.2), 1.465 (br t, 8H, J=6.5, CH.sub.2), 1.556 (br t, 8H,
J=6.5, CH.sub.2), 2.688 (s, 4H, CH.sub.2), 2.720 (t, 8H, J=6.0,
CH.sub.2), 3.132 (q, 8H, J=6.5, CH.sub.2), 3.366 (q, 8H, J=6.5,
CH.sub.2), 3.460 (q, 8H, J=6.0, CH.sub.2), 3.765 (s, 12H, NH),
5.041 (s, 8H, OCH.sub.2), 5.096 (s, 8H, NH), 7.073 (t, 4H, J=7.5,
ArH), 7.26-7.38 (m, 20H, ArH), 7.517 (t, 4H, J=5.7, NH), 7.675 (d,
4H, J=7.5, ArH), 7.812 (d, 4H, J=7.5, ArH), 7.857 (t, 4H, J=5.5,
NH). .sup.13 C NMR (500 MHz, CDCl.sub.3, 25.degree. C.)
.delta.26.22, 26.51, 29.31, 29.78, 37.72, 39.70, 40.78, 51.88,
53.40, 63.17, 66.40, 124.61, 127.34, 127.92, 127.96, 129.03,
133.69, 136.59, 155.53, 156.43, 165.44. 11.3
H22tetra(6-amino-1-hexaneamido)IAM, 23
22 (0.25 g, 0.13 mmol) was dissolved in dry degassed CH.sub.2
Cl.sub.2 (20 mL). The solution was cooled in an ice bath and
BBr.sub.3 (0.5 mL, 5.7 mmol) was added via syringe under nitrogen.
The resulted pale yellow slurry was stirred for 48 hrs, after which
the volatile was removed under vacuum and the residue quenched with
methanol (20 mL). The methanol solution was diluted with water 40
mL) and boiled until a colorless transparent solution was obtained
after reduction of the volume to 10 mL. The solution was cooled,
and the compound appeared as a gel that was separated from water by
centrifugation. After drying (vacuum oven overnight) 234 mg of
white solid was collected. (+)-FABMS: m/Z: 1281.7 [MH.sup.+ ].
.sup.1 H NMR (500 MHz, DMSO-d.sub.6), 25.degree. C.) .delta.1.310
(br s, 16H, CH.sub.2), 1.541 (br s, 16H, CH.sub.2), 2.731 (q, 8H,
J=6.0, CH.sub.2), 3.277 (q, 8H, J=6.0, CH.sub.2), 3.468 (br s, 12H,
CH.sub.2), 3.764 (br s, 8H, CH.sub.2), 6.922 (t, 4H, J=7.8, ArH),
7.968 (s, 4H, NH), 8.019 (d, J=7.8, 4H, ArH), 8.084 (d, J=7.8, 4H,
ArH), 8.993 (br s, 4, NH). 11.4 Tb[H(2,2)
tetra(6-amino-1-hexaneamido)IAM], 24
23 (0.042 mmol) was suspended in 12 mL of a solution of MeOH
containing Tb(NO.sub.3).sub.3.6H.sub.2 O (0.042 mmol, 99.999%). The
suspension was heated until reflux and the solid dissolved. 45
drops of collidine were added to the resulting solution. A strong
green emission of the solution was observed upon UV irradiation.
After 13 hours of reflux, the solvent was evaporated until the
precipitation of a white solid. The product was filtered and washed
with Et.sub.2 O. After drying of the product (vacuum oven), 67 mg
of product was collected. Calcd. (Found) for TbC.sub.66 H.sub.96
N.sub.14 O.sub.12 (NO.sub.3)(HBr).sub.7.9H.sub.2 O: C, 35.65
(35.60); H, 5.44 (5.48); N, 9.45 (9.43).
Example 12
Example 12 illustrates the synthesis of compounds of the invention
having backbones of variable length. The synthetic scheme is set
forth in FIG. 21. 12.1 Synthesis of
2-Methoxy-1-(2-mercaptothiazolide)isophthalamide methylamide,
44
To a solution of 1 (20 g, 0.050 mol) in methylene chloride (200
mL), a mixture of 2 mL methylamine solution (40% wt in water, d
=0.902) and 50 mL isopropanol was added dropwise over 8 h. The
reaction mixture was evaporated to dryness and the residue was
purified by a gradient flash silica column (1-5% methanol in
methylene chloride). 5.8 g of the desired product were obtained as
a yellow crystalline solid. Yield 81% based on the methylamine. 7.8
g of unreacted dithiazolide were also recovered during the
separation.
.sup.1 H NMR (500 MHz, CDCl.sub.3, 25.degree. C.) (FIG. 14)
.delta.: 3.012 (d, J=5.0, 3H, NHCH.sub.3), 3.449 (t, J=7.5, 2H,
CH.sub.2), 3.866 (s, 3H, OCH.sub.3), 4.660 (t, J=7.5, 2H,
CH.sub.2), 7.227 (t, J=7.5, 1H, ArH), 7.416 (d, J=7.5, 1H, ArH),
7.425 (s, br, 1H, Amide H), 8.124 (d, J=7.5, 1H, ArH). .sup.13 C
NMR (500 MHz, CDCl.sub.3, 25.degree. C.) (FIG. 15) .delta.: 26.30,
28.73, 55.32, 62.62, 123.81, 126.78, 128.70, 131.49, 155.06,
164.98, 201.15. 12.2 Synthesis of Me.sub.4 H(3,2)IAM, 45
To a solution of H(3,2)-amine (1 mmol) in CH.sub.2 Cl.sub.2 (50
mL), compound 44 (4.8 mmol) was added. The mixture was stirred
until TLC reveal that the reaction was ended. The reaction mixture
was purified trough a gradient flash silica gel column (2-7%
CH.sub.3 OH in CH.sub.2 Cl.sub.2). The pure product was obtained as
a white foam. Yield 81%.
(+)-FABMS: m/Z: 1011.8 [MH.sup.30 ]. .sup.1 H NMR (500 MHz,
CDCl.sub.3, 25.degree. C.), .delta.1.624 (quint, J=6.5, 2H,
CH.sub.2), 2.524 (t, J=6.5, 4H, CH.sub.2), 2.649 (t, 8H, J=6.5,
CH.sub.2), 2.935 (d, 12H, J=5.0, CH.sub.2), 3.448 (q, J=6.5, 8H,
CH.sub.2), 3.777 (s, 12H, CH.sub.3) 7.097 (t, 4H, J=7.5, ArH),
7.451 (t, 4H, J=5.5, Amide H), 7.714 (d, 4H, J=7.5, ArH), 7.813 (t,
4H, J=5.5, Amide H), 7.856 (d, J=7.5, 4H, ArH). .sup.13 C NMR (500
MHz, CDCl.sub.3, 25.degree. C.), .delta.: 24.15, 26.67, 37.83,
52.06, 53.21, 63.09, 124.54, 127.53, 128.41, 133.23, 133.63,
155.60, 165.46, 165.99. 12.3 Synthesis of H.sub.4 H(3,2)IAM, 47
45 (0.5 mmol) was dissolved in dry degassed CH.sub.2 Cl.sub.2 (20
mL) and the resulting solution was cooled in an ice bath. BBr.sub.3
(11.4 mmol) was added via syringe under nitrogen. The resulted pale
yellow slurry was stirred for 48 hrs. The volatile was then removed
under vacuum and the residue quenched with methanol (30 mL). The
methanol solution was diluted with water (40 mL) and boiled until a
colorless transparent solution was obtained and its volume was
reduced to 10 mL. The solution was cooled and a white precipitate
appeared, which was collected by filtration and dried under vacuum.
Yield 62%.
(+)-FABMS: m/Z: 1874.9 [MH.sup.+ ]. .sup.1 H NMR (500 MHz, CD.sub.3
OD, 25.degree. C.), .delta.2.467 (quint, J=6.0, 2H, CH.sub.2),
2.871 (s, 12H, CH.sub.3), 3.625 (t, 8H, J=6.0, CH.sub.2), 3.65-3.75
(m, 8H, CH.sub.2), 3.85-3.95 (m, 4H, CH.sub.2), 6.734 (t, 4H,
J=7.5, ArH), 7.678 (d, 4H, J=7.5, ArH), 7.772 (t, 4H, J=7.5, ArH).
12.4 Synthesis of Me.sub.4 H(4,2)IAM, 46
This compound was prepared by using the same procedure as described
for compound 45 except H(4,2)-amine being used instead of
H(3,2)-amnine. Yield 84%.
(+)-FABMS: m/Z: 1025.9 [MH.sup.+ ]. .sup.1 H NMR (500 MHz,
CDCl.sub.3, 25.degree. C.), .delta.1.412 (s,br, 4H, CH.sub.2),
2.490 (s,br, 4H, CH.sub.2), 2.463 (t, 8H, J=6.0, CH.sub.2), 2.945
(d, 12H, J=5.0, CH.sub.2), 3.464 (q, J=6.5, 8H, CH.sub.2), 3.767
(s, 12H, CH.sub.3), 7.111 (t, 4H, J=7.5, ArH), 7.396 (q, 4H, J=5.0,
Amide H), 7.752 (d, 4H, J=7.5, ArH), 7.795 (t, 4H, J=5.0, Amide H),
7.839 (d, J=7.5, 4H, ArH). .sup.13 C NMR (500 MHz, CDCl.sub.3,
25.degree. C.), .delta.: 24.01, 26.23, 37.64, 49.72, 52.58, 52.41,
53.64, 62.65, 124.03, 127.29, 128.00, 132.79, 133.00, 155.29,
165.20, 165.87. 12.5 Synthesis of H.sub.4 H(4,2)IAM, 48
This compound was deprotected by the classical BBr.sub.3
deprotection procedure described for compound 47. The pure material
was collected as a white solid. Yield 71%.
(+)-FABMS: m/Z: 969.7 [MH.sup.+ ]. .sup.1 H NMR (500 MHz,
DMSO-d.sub.6, 25.degree. C.), .delta.1.766 (s,br, 4H, CH.sub.2),
2.812 (d, J=5.0, 12H, CH.sub.3), 3.31 (s,br, 4H, CH.sub.2), 3.386
(s,br, 8H, CH.sub.2), 3.714 (d,br, J=5.5, 8H, CH.sub.2), 6.948 (t,
4H, J=7.5, ArH), 7.984 (d, 8H, J=7.5, ArH), 8.844 (t, J=5.5, 4H,
AmideH), 8.984 (d,br, J=5.5, 4H, AmideH), 9.549 (s,br, 2H,
phenolH). .sup.1 H NMR (500 MHz, D.sub.2 O--NaOD, 25.degree. C.),
.delta.1.313 (s,br, 4H, CH.sub.2), 2.388 (s,br, 4H, CH.sub.2),
2.593 (t, J=6.0, 8H, CH.sub.2), 2.755 (s,br, 12H, CH.sub.3), 3.343
(t, J=6.0, 8H, CH.sub.2), 6.477 (t, 4H, J=7.5, ArH), 7.816 (d, 8H,
J=7.5, ArH).
It is to be understood that the above description is intended to be
illustrative and not restrictive. Many embodiments will be apparent
to those of skill in the art upon reading the above description.
The scope of the invention should, therefore, be determined not
with reference to the above description, but should instead be
determined with reference to the appended claims, along with the
full scope of equivalents to which such claims are entitled. The
disclosures of all articles and references, including patent
applications and publications are incorporated herein by
reference.
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